Prevention and treatment of acute renal failure and other kidney diseases by inhibition of p53 by siRNA

ABSTRACT

The invention relates to a double-stranded compound, preferably an oligoribonucleotide, which down-regulates the expression of a human p53 gene. The invention also relates to a pharmaceutical composition comprising the compound, or a vector capable of expressing the oligoribonucleotide compound, and a pharmaceutically acceptable carrier. The present invention also contemplates a method of treating a patient suffering from acute renal failure or other kidney diseases comprising administering to the patient the pharmaceutical composition in a therapeutically effective dose so as to thereby treat the patient.

This application is a continuation-in-part of U.S. Ser. No. 11/655,610filed Jan. 18, 2007 and claims the benefit of U.S. ProvisionalApplication No. 60/781,037, filed Mar. 9, 2006 and of U.S. ProvisionalApplication No. 60/854,503, filed Oct. 25, 2006, all of which are herebyincorporated by reference in their entirety.

Throughout this application various patent and scientific publicationsare cited. The disclosures for these publications in their entiretiesare hereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION siRNAs and RNA Interference

RNA interference (RNAi) is a phenomenon involving double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing. Originally,attempts to study this phenomenon and to manipulate mammalian cellsexperimentally were frustrated by an active, non-specific antiviraldefence mechanism which was activated in response to long dsRNAmolecules; see Gil et al. 2000, Apoptosis, 5:107-114. Later it wasdiscovered that synthetic duplexes of 21 nucleotide RNAs could mediategene specific RNAi in mammalian cells, without the stimulation of thegeneric antiviral defence mechanisms (see Elbashir et al. Nature 2001,411:494-498 and Caplen et al. Proc Natl Acad Sci 2001, 98:9742-9747). Asa result, small interfering RNAs (siRNAs), which are shortdouble-stranded RNAs, have become powerful tools in attempting tounderstand gene function.

Thus, RNA interference (RNAi) refers to the process of sequence-specificpost-transcriptional gene silencing in mammals mediated by smallinterfering RNAs (siRNAs) (Fire et al, 1998, Nature 391, 806) ormicroRNAs (miRNAs) (Ambros V. Nature 431:7006,350-355(2004); and BartelD P. Cell. 2004 Jan. 23; 116(2): 281-97 MicroRNAs: genomics, biogenesis,mechanism, and function). The corresponding process in plants iscommonly referred to as specific post-transcriptional gene silencing orRNA silencing and is also referred to as quelling in fungi. An siRNA isa double-stranded RNA molecule which down-regulates or silences(prevents) the expression of a gene/mRNA of its endogenous (cellular)counterpart. RNA interference is based on the ability of dsRNA speciesto enter a specific protein complex, where it is then targeted to thecomplementary cellular RNA and specifically degrades it. Thus, the RNAinterference response features an endonuclease complex containing ansiRNA, commonly referred to as an RNA-induced silencing complex (RISC),which mediates cleavage of single-stranded RNA having a sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA may take place in the middle of the region complementaryto the antisense strand of the siRNA duplex (Elbashir et al 2001, GenesDev., 15, 188). In more detail, longer dsRNAs are digested into short(17-29 bp) dsRNA fragments (also referred to as short inhibitoryRNAs—“siRNAs”) by type III RNAses (DICER, DROSHA, etc., Bernstein etal., Nature, 2001, v.409, p. 363-6; Lee et al., Nature, 2003, 425, p.415-9). The RISC protein complex recognizes these fragments andcomplementary mRNA. The whole process is culminated by endonucleasecleavage of target mRNA (McManus&Sharp, Nature Rev Genet, 2002, v.3, p.737-47; Paddison &Hannon, Curr Opin Mol Ther. 2003 June; 5(3): 217-24).For information on these terms and proposed mechanisms, see BernsteinE., Denli A M. Hannon G J: 2001 The rest is silence. RNA. I; 7(11):1509-21; Nishikura K.: 2001 A short primer on RNAi: RNA-directed RNApolymerase acts as a key catalyst. Cell. I 16; 107(4): 415-8 and PCTpublication WO 01/36646 (Glover et al).

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; see for example Chalk A M, Wahlestedt C,Sonnhammer E L. 2004 Improved and automated prediction of effectivesiRNA Biochem. Biophys. Res. Commun. June 18; 319(1): 264-74; Sioud M,Leirdal M., 2004, Potential design rules and enzymatic synthesis ofsiRNAs, Methods Mol Biol.; 252:457-69; Levenkova N, Gu Q, Rux J J. 2004,Gene specific siRNA selector Bioinformatics. I 12; 20(3): 430-2. andUi-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A,Ueda R, Saigo K., Guidelines for the selection of highly effective siRNAsequences for mammalian and chick RNA interference Nucleic Acids Res.2004 I 9; 32(3):936-48. Se also Liu Y, Braasch D A, Nulf C J, Corey D R.Efficient and isoform-selective inhibition of cellular gene expressionby peptide nucleic acids, Biochemistry, 2004 I 24; 43(7):1921-7. Seealso PCT publications WO 2004/015107 (Atugen) and WO 02/44321 (Tuschl etal), and also Chiu Y L, Rana T M. siRNA function in RNAi: a chemicalmodification analysis, RNA 2003 September; 9(9):1034-48 and U.S. Pat.Nos. 5,898,031 and 6,107,094 (Crooke) for production of modified/morestable siRNAs.

Several groups have described the development of DNA-based vectorscapable of generating siRNA within cells. The method generally involvestranscription of short hairpin RNAs that are efficiently processed toform siRNAs within cells. Paddison et al. PNAS 2002, 99:1443-1448;Paddison et al. Genes & Dev 2002, 16:948-958; Sui et al. PNAS 2002,8:5515-5520; and Brummelkamp et al. Science 2002, 296:550-553. Thesereports describe methods to generate siRNAs capable of specificallytargeting numerous endogenously and exogenously expressed genes.

Several studies have revealed that siRNA therapeutics are effective invivo in both mammals and in humans. Bitko et al., have shown thatspecific siRNA molecules directed against the respiratory syncytialvirus (RSV) nucleocapsid N gene are effective in treating mice whenadministered intranasally (Bitko et al., “Inhibition of respiratoryviruses by nasally administered siRNA”, Nat. Med. 2005, 11(1):50-55). Areview of the use of siRNA in medicine was recently published by BarikS. in J. Mol. Med (2005) 83: 764-773). Furthermore, a phase I clinicalstudy with short siRNA molecule that targets the VEGFR1 receptor for thetreatment of Age-Related Macular Degeneration (AMD) has been conductedin human patients. The siRNA drug administered by an intravitrealinter-ocular injection was found effective and safe in 14 patientstested after a maximum of 157 days of follow up (Boston Globe Jan. 21,2005).

The p⁵³ Gene and Polypeptide

The human p53 gene is a well-known and highly studied gene. The p53polypeptide plays a key role in cellular stress response mechanisms byconverting a variety of different stimuli, for example DNA damagingconditions, such as gamma-irradiation, deregulation of transcription orreplication, and oncogene transformation, into cell growth arrest orapoptosis (Gottlieb et al, 1996, Biochem. Biophys. Acta, 1287, p. 77).The p53 polypeptide is essential for the induction of programmed celldeath or “apoptosis” as a response to such stimuli.

Most anti-cancer therapies damage or kill also normal cells that containnative p53, causing severe side effects associated with the damage ordeath of healthy cells. Since such side effects are to a great extentdetermined by p53-mediated death of normal cells, the temporarysuppression of p53 during the acute phase of anti-cancer therapy hasbeen suggested as a therapeutic strategy to avoid these severe toxicevents. This was described in U.S. Pat. No. 6,593,353 and in Komarov P Get al, 1999, A chemical inhibitor of p53 that protects mice from theside effects of cancer therapy, Science, 285(5434):1651, 1653. p53 hasbeen shown to be involved in chemotherapy and radiation-inducedalopecia. (Botcharev et al, 2000, p53 is essential forChemotherapy-induced Hair Loss, Cancer Research, 60, 5002-5006).

Chemical-Induced Ototoxicity

The toxic effects of various ototoxic therapeutic drugs on auditorycells and spiral ganglion neurons are often the limiting factor fortheir therapeutic usefulness. Main ototoxic drugs include the widelyused chemotherapeutic agent cisplatin and its analogs, commonly usedaminoglycoside antibiotics, e.g. gentamicin, for the treatment ofinfections caused by gram-negative bacteria, quinine and its analogs,salicylate and its analogs, and loop-diuretics.

For example, antibacterial aminoglycosides such as gentamicins,streptomycins, kanamycins, tobramycins, and the like are known to haveserious toxicity, particularly ototoxicity and nephrotoxicity, whichreduces the usefulness of such antimicrobial agents (see Goodman andGilman's The Pharmacological Basis of Therapeutics, 6th ed., A. GoodmanGilman et al., eds; Macmillan Publishing Co., Inc., New York, pp.1169-71 (1980)). Clearly, ototoxicity is a dose-limiting side-effect ofantibiotic administration. From 4 to 15% of patients receiving 1 gramper day for greater than 1 week develop measurable hearing loss, whichslowly becomes worse and can lead to complete permanent deafness iftreatment continues.

Ototoxicity is also a serious dose-limiting side-effect for cisplatin, aplatinum coordination complex, that has proven effective on a variety ofhuman cancers including testicular, ovarian, bladder, and head and neckcancer. Cisplatin (Platinol®) damages auditory and vestibular systems.Salicylates, such as aspirin, are the most commonly used therapeuticdrugs for their anti-inflammatory, analgesic, anti-pyretic andanti-thrombotic effects. Unfortunately, they too have ototoxic sideeffects. They often lead to tinnitus (“ringing in the ears”) andtemporary hearing loss. Moreover, if the drug is used at high doses fora prolonged time, the hearing impairment can become persistent andirreversible.

Accordingly, there exists a need for means to prevent, reduce or treatthe incidence and/or severity of inner ear disorders and hearingimpairments involving inner ear tissue, particularly inner ear haircells. Of particular interest are those conditions arising as anunwanted side-effect of ototoxic therapeutic drugs including cisplatinand its analogs, aminoglycoside antibiotics, salicylate and its analogs,or loop diuretics. In addition, there exits a need for methods whichwill allow higher and thus more effective dosing with theseototoxicity-inducing pharmaceutical drugs, while concomitantlypreventing or reducing ototoxic effects caused by these drugs. What isneeded is a method that provides a safe, effective, and prolonged meansfor prophylactic or curative treatment of hearing impairments related toinner ear tissue damage, loss, or degeneration, particularlyototoxin-induced and particularly involving inner ear hair cells.

Without being bound by theory, it is believed that cisplatin drugs andother drugs that induce ototoxicity (such as aminoglycoside antibiotics)may induce the ototoxic effects via programmed cell death or apoptosisin inner ear tissue, particularly inner ear hair cells (Zhang et al.,Neuroscience 120 (2003) 191-205; Wang et al., J. Neuroscience23((24):8596-8607). In mammals, auditory hair cells are produced onlyduring embryonic development and do not regenerate if lost duringpostnatal life, therefore, a loss of hair cells will result in profoundand irreversible deafness. Unfortunately, at present, there are noeffective therapies to treat the cochlea and reverse this condition.Thus, an effective therapy to prevent cell death of auditory hair cellswould be of great therapeutic value.

Acute Renal Failure (ARF).

ARF is a clinical syndrome characterized by rapid deterioration of renalfunction that occurs within days. Without being bound by theory acutekidney injury may be the result of renal ischemia-reperfusion injury(IRI) that occurs, for example, in patients undergoing major surgerysuch as major cardiac surgery.

The principal feature of ARF is an abrupt decline in glomerularfiltration rate (GFR), resulting in the retention of nitrogenous wastes(urea, creatinine). In the general world population 170-200 cases ofsevere ARF per million population occur annually. To date, there is nospecific treatment for ARF. Several drugs have been found to amelioratetoxic and ischemic experimental ARF, as manifested by lower serumcreatinine levels, reduced histological damage and faster recovery ofrenal function in different animal models. These include anti-oxidants,calcium channel blockers, diuretics, vasoactive substances, growthfactors, anti-inflammatory agents and more. However, those drugs thathave been studied in clinical trials showed no benefit, and their use inARF has not been approved.

In the majority of hospitalized patients, ARF is caused by acute tubularnecrosis (ATN), which results from ischemic and/or nephrotoxic insults.Renal hypoperfusion is caused by hypovolemic, cardiogenic and septicshock, by administration of vasoconstrictive drugs or renovascularinjury. Nephrotoxins include exogenous toxins such as contrast media andaminoglycosides as well as endogenous toxin such as myoglobin. Recentstudies, however, indicate that apoptosis in renal tissue is prominentin most human cases of ARF, with the principal site of apoptotic celldeath being the distal nephron. During the initial phase of ischemicinjury, loss of integrity of the actin cytoskeleton leads to flatteningof the epithelium, with loss of the brush border, loss of focal cellcontacts, and subsequent disengagement of the cell from the underlyingsubstratum. It has been suggested that apoptotic tubule cell death maybe more predictive of functional changes than necrotic cell death(Komarov et al. Science. 1999. 285(5434):1733-7); see also (Supavekin etal. Kidney Int. 2003. 63(5):1714-24).

Hamar et al., administered siRNA targeting Fas, a mediator of apoptosis,to mice by hydrodynamic injection or via renal vein infusion (PNAS 2004.102(41):14883-14888).

Currently there are no satisfactory modes of therapy for the preventionand/or treatment of acute renal failure and related kidney diseases anddisorders.

SUMMARY OF THE INVENTION

The present invention provides double stranded oligoribonucleotides thatinhibit the p53 gene. The invention also provides a pharmaceuticalcomposition comprising one or more such oligoribonucleotides, and avector capable of expressing the oligoribonucleotide. The presentinvention also relates to methods and compositions for treating orpreventing acute renal failure (ARF) following ischemic-reperfusionevent, wherein the composition is administered in a therapeuticallyeffective dose following the initiation of the ischemic-reperfusionevent. The present invention also relates to methods and compositionsfor treating or preventing the incidence or severity of hearingimpairment (or balance impairment), particularly hearing impairmentassociated with cell death of the inner ear hair cells or outer ear haircells. The methods and compositions involve administering to a mammal inneed of such treatment a prophylactically or therapeutically effectiveamount of one or more compounds which down-regulate expression of thep53 gene, particularly small interfering RNAs (siRNAs), small moleculeinhibitors of p53 as described herein or antibodies to p53 polypeptide.

In one embodiment, the siRNA molecules disclosed herein may be used inthe treatment of acute renal failure (ARF), which is characterized byrapid deterioration of renal function associated with apoptotic celldeath in the renal tissue. In some embodiments kidney injury isischemic-reperfusion injury resulting from major surgery. In variousembodiments major surgery is cardiac surgery. In other embodimentssurgery is transplant surgery. In certain preferred embodiments kidneyinjury is acute injury. In some embodiments kidney injury results fromsepsis or chemical-induced nephrotoxicity.

According to one aspect the present invention provides a method oftreating a patient suffering from a kidney injury following anischemic-reperfusion event comprising administering to the patient acomposition comprising one or more compounds having the structure:

-   -   5′ (N)_(x)-Z 3′ (antisense strand)    -   3′ Z′-(N′)_(y) 5′ (sense strand)

wherein each of N and N′ is a ribonucleotide which may be modified orunmodified in its sugar residue;

wherein each of (N)_(x) and (N′)_(y) is an oligomer in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;

wherein each of x and y is an integer between 18 and 40;

wherein each of Z and Z′ may be present or absent, but if present is 1-5consecutive nucleotides covalently attached at the 3′ terminus of thestrand in which it is present;

wherein the sequence of (N′)_(y) is present within a mRNA whose sequenceis set forth in SEQ ID NO: 1, and wherein the composition isadministered in a therapeutically effective dose following theinitiation of the ischemic-reperfusion event so as to thereby treat thepatient.

In some embodiments each of (N)_(x) and (N′)_(y) are selected from anoliogomer set forth in any one of SEQ ID NOS: 3-387.

In another aspect the present invention provides a compound having thestructure:

-   -   5′ (N)_(x)-Z 3′ (antisense strand)    -   3′ Z′-(N′)_(y) 5′ (sense strand)

wherein each of N and N′ is a nucleotide which may be modified orunmodified in its sugar residue;

wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond andwherein each of (N)_(x) and (N′)_(y) are selected from SEQ ID NOS:317-387;

wherein each of x and y is an integer between 18 and 40;

wherein each of Z and Z′ may be present or absent, but if present is 1-5consecutive nucleotides covalently attached at the 3′ terminus of thestrand in which it is present.

In some embodiments the covalent bond joining each consecutive N or N′is a phosphodiester bond. In various embodiments all the covalent bondsare phosphodiester bonds.

In some embodiments the sequence of (N)_(x) and (N′)_(y) are fullycomplementary.

In some embodiments the compound is blunt ended, for example whereinboth Z and Z′ are absent. In an alternative embodiment, the compoundcomprises at least one 3′ overhang, wherein at least one of Z or Z′ ispresent. Z and Z′ can independently comprise one or more covalentlylinked modified or non-modified nucleotides, for example inverted dT ordA; dT, LNA, mirror nucleotide and the like. In some embodiments each ofZ and Z′ are independently selected from dT and dTdT.

In some embodiments N or N′ comprises a modification in the sugarresidue of one or more ribonucleotides. In other embodiments thecompound comprises at least one ribonucleotide modified in the sugarresidue. In some embodiments the compound comprises a modification atthe 2′ position of the sugar residue. In some embodiments themodification in the 2′ position comprises the presence of an amino, afluoro, an alkoxy or an alkyl moiety. In certain embodiments the 2′modification comprises a methoxy moiety. A presently preferredmodification is a 2′ methoxy of the sugar residue (2′-O-methyl; 2′-O-Me;2′-O—CH₃).

In various embodiments the compound comprises an antisense sequencepresent in Tables A, B, C and C2 (SEQ ID NOS:3-387). In otherembodiments the present invention provides a mammalian expression vectorcomprising an antisense sequence present in Tables A, B, C and C2 (SEQID NOS:3-387).

In another aspect the present invention provides a pharmaceuticalcomposition comprising a novel compound of the invention; and apharmacologically acceptable carrier or excipient.

In yet another aspect the present invention provides a method for thetreatment of a subject having acute renal failure (ARF) or relateddisease or disorder comprising the step of administering to the subjectan amount of an siRNA which reduces or inhibits expression of p53 (SEQID NO:2), wherein the composition is administered in a therapeuticallyeffective dose following the initiation of the ischemic-reperfusionevent so as to thereby treat the patient.

In another embodiment the present invention provides methods andcompositions for treating a patient suffering from hearing impairment,or other oto-pathologies associated with cell death of inner ear haircells or outer ear hair cells. Such oto-pathologies may be the result ofacoustic trauma, mechanical trauma, or ototoxin-induced hearing loss.The methods of the invention comprising administering to the patient oneor more compounds which down-regulate expression of the p53 gene,particularly siRNAs that inhibit p53, typically as a pharmaceuticalcomposition, in a therapeutically effective dose so as to thereby treatthe patient. Since long-term p53 inactivation may significantly increasethe risk of cancer, it is preferred that the inhibition of p53 using themolecules of the present invention be temporary or local.

In one embodiment, the present invention provides for improvedcompositions and methods for treatments requiring administration of apharmaceutical drug having an ototoxic, hearing-impairing side-effect,in combination with a therapeutically effective amount of one or moresiRNA molecules that inhibit p53, to treat or prevent the ototoxicityinduced by the pharmaceutical drug. The compositions of the inventioncan be administered at a suitable interval(s) either prior to,subsequent to, or substantially concurrently with the administration ofthe ototoxic, hearing-impairing drug that induces inner ear apoptotictissue damage.

Accordingly, it is an object of the invention to provide an improvedcomposition containing a therapeutically effective amount of one or moresiRNA molecules that inhibit p53 in combination with an ototoxic,hearing-impairing pharmaceutical drug for administration to a mammal.Preferably, the combination drugs are administered separately; the siRNAmolecules that inhibit p53 are administered locally while the ototoxic,hearing-impairing pharmaceutical drug is administered systemically. ThesiRNA molecules may be administered prior to, simultaneously with orsubsequent to the ototoxic drug. Such combination compositions canfurther contain a pharmaceutically acceptable carrier. Thepharmaceutical composition will have lower ototoxicity than the ototoxicpharmaceutical alone, and, preferably, will have a higher dosage of theototoxic pharmaceutical than typically used. Examples of such improvedcompositions include cisplatin or other ototoxic neoplastic agent or anaminoglycoside antibiotic(s) in combination with the therapeuticallyeffective amount of one or more siRNA molecules that inhibit p53.

Still further, the invention relates to the use of the compositions ofthe invention in cases where diuretics are needed. The present inventionprovides a solution to the art that has long sought a therapy and amedicament which can treat the ototoxic effects currently associatedwith certain diuretics, and particular with the more popular andcommonly used loop-diuretics, without sacrificing their diureticeffectiveness.

Still further, the invention relates to the use of the compositions ofthe invention in cases where quinine or quinine-like compounds areneeded. The present invention provides a solution to the art that haslong sought a therapy and a medicament which can treat the ototoxiceffects currently associated with certain quinines without sacrificingtheir effectiveness.

Still further, the invention relates to a method for treating orpreventing the incidence or severity of hearing impairment in a patientcomprising administering to the patient a composition comprising aneffective amount of naked siRNA molecules. Preferably, the naked siRNAmolecules are applied directly to the round window membrane of thecochlea or administered by transtympanic injection. Further, the nakedsiRNA molecules are preferably directed against at least onepro-apoptotic gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. This figure represents the nucleotide sequence of the human p53gene—SEQ ID NO: 1.

FIG. 2. This figure represents the amino acid sequence of the human p53polypeptide—SEQ ID NO:2.

FIG. 3. This figure shows Western Blot results demonstrating the effectof various human p53 siRNAs on p53 expression.

FIG. 4. This figure shows Western Blot results demonstrating the effectof various mouse p53 siRNAs on p53 expression.

FIG. 5. This figure shows the effect of p53 siRNA treatment onacoustic-induced hair cell death in the cochlea of chinchilla.

FIG. 6. This figure shows the level of serum creatinine as an indicationfor acute renal failure in animals that underwent bilateral kidneyarterial clamp and were treated with p53 siRNA compound or a control, asindicated.

FIG. 7. This figure shows the extent of tubular necrosis in renal tissuein animals that underwent bilateral kidney arterial clamp and weretreated with the p53 siRNA compound.

FIG. 8. This figure demonstrates that p53 siRNA treatment down-regulatedthe expression of the pro-apoptotic gene Puma in the corticalcompartment of the kidney in animal subjected to ischemia-reperfusionkidney injury.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to compounds which down-regulateexpression of the p53 gene, particularly to novel small interfering RNAs(siRNAs), and to the use of these novel siRNAs in the treatment ofvarious diseases and medical conditions in particular various forms ofacute renal failure or hearing impairment as described above. Preferredlists of such siRNA are in Tables A, B, C and C2.

The inventors of the present invention have found that it is beneficialto induce temporary inhibition of p53 in order to treat any of the abovediseases or disorders. Methods, molecules and compositions which inhibitp53 are discussed herein at length, and any of said molecules and/orcompositions may be beneficially employed in the treatment of a patientsuffering from any of said conditions.

The present invention provides methods and compositions for inhibitingexpression of a target p53 gene in vivo. In general, the method includesadministering oligoribonucleotides, such as small interfering RNAs(i.e., siRNAs) that are targeted to a particular p53 gene mRNA andhybridize to, or interact with, the mRNAs under biological conditions(within the cell), or a nucleic acid material that can produce siRNA ina cell, in an amount sufficient to down-regulate expression of a targetgene by an RNA interference mechanism. In particular, the subject methodcan be used to inhibit expression of the p53 gene for treatment of adisease.

In accordance with the present invention, the siRNA molecules orinhibitors of the p53 gene may be used as drugs to treat variouspathologies accompanied by an elevated level of p53 polypeptide. Sincelong-term p53 inactivation can significantly increase the risk ofcancer, it is preferred that the inhibition of p53 using the moleculesof the present invention be temporary/reversible.

The present invention provides double-stranded oligoribonucleotides(siRNAs), which down-regulate the expression of the p53 gene. An siRNAof the invention is a duplex oligoribonucleotide in which the sensestrand is derived from the mRNA sequence of the p53 gene, and theantisense strand is complementary to the sense strand. In general, somedeviation from the target mRNA sequence is tolerated withoutcompromising the siRNA activity (see e.g. Czauderna et al 2003 NucleicAcids Research 31(11), 2705-2716). An siRNA of the invention inhibitsgene expression on a post-transcriptional level with or withoutdestroying the mRNA. Without being bound by theory, siRNA may target themRNA for specific cleavage and degradation and/or may inhibittranslation from the targeted message.

There are at least four variant p53 polypeptides (see Bourdon et al.Genes Dev. 2005; 19: 2122-2137). The sequence given in FIG. 1 is thenucleotide sequence of gi-8400737. The corresponding polypeptidesequence has 393 amino acids; see FIG. 2. All variants and any othersimilar minor variants are included in the definition of p53 polypeptideand in the definition of the p53 genes encoding them.

As used herein, the term “p53 gene” is defined as any homolog of the p53gene having preferably 90% homology, more preferably 95% homology, andeven more preferably 98% homology to the amino acid encoding region ofSEQ ID NO: 1 or nucleic acid sequences which bind to the p53 gene underconditions of highly stringent hybridization, which are well-known inthe art (for example, see Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1988), updated in 1995 and1998.

As used herein, the term “p53”, or “p53 polypeptide” is defined as anyhomolog of the p53 polypeptide having preferably 90% homology, morepreferably 95% homology, and even more preferably 98% homology to SEQ IDNO:2, as either full-length or a fragment or a domain thereof, as amutant or the polypeptide encoded by a spliced variant nucleic acidsequence, as a chimera with other polypeptides, provided that any of theabove has the same or substantially the same biological function as thep53 polypeptide.

As used herein, an “inhibitor” is a compound which is capable ofinhibiting or reducing the expression or activity of a gene or theproduct of such gene to an extent sufficient to achieve a desiredbiological or physiological effect. The term “inhibitor” as used hereinrefers to one or more of an oligonucleotide inhibitor, including siRNA,shRNA, aptamers, antisense molecules, miRNA and ribozymes, as well asantibodies.

As used herein, the term “Oligonucleotide” refers to a sequence havingfrom about 2 to about 50 linked nucleotides or linked modifiednucleotides, or a combination of modified and unmodified nucleotide.Oligonucleotide includes the terms oligomer, antisense strand and sensestrand.

“Nucleotide” is meant to encompass deoxyribonucleotides andribonucleotides, which may be natural or synthetic, and or modified orunmodified. Modifications include changes to the sugar moiety, the basemoiety and or the linkages between ribonucleotides in theoligoribonucleotide.

Generally, the siRNAs used in the present invention comprise aribonucleic acid comprising a double stranded structure, whereby thedouble-stranded structure comprises a first strand and a second strand,whereby the first strand comprises a first stretch of contiguousnucleotides and whereby said first stretch is at least partiallycomplementary to a target nucleic acid, and the second strand comprisesa second stretch of contiguous nucleotides and whereby said secondstretch is at least partially identical to a target nucleic acid,whereby said first strand and/or said second strand comprises aplurality of groups of modified nucleotides having a modification at the2′-position whereby within the strand each group of modified nucleotidesis flanked on one or both sides by a flanking group of nucleotideswhereby the flanking nucleotides forming the flanking group ofnucleotides is either an unmodified nucleotide or a nucleotide having amodification different from the modification of the modifiednucleotides. Further, said first strand and/or said second strand maycomprise said plurality of modified nucleotides and may comprises saidplurality of groups of modified nucleotides.

The group of modified nucleotides and/or the group of flankingnucleotides may comprise a number of nucleotides whereby the number isselected from the group comprising one nucleotide to 10 nucleotides. Inconnection with any ranges specified herein it is to be understood thateach range discloses any individual integer between the respectivefigures used to define the range including said two figures definingsaid range. In the present case the group thus comprises one nucleotide,two nucleotides, three nucleotides, four nucleotides, five nucleotides,six nucleotides, seven nucleotides, eight nucleotides, nine nucleotidesand ten nucleotides.

The groups of modified nucleotides and flanking nucleotides may beorganized in a pattern on at least one of the strands. In someembodiments the first and second strands comprise a pattern of modifiednucleotides. In various embodiments the pattern of modified nucleotidesof said first strand is identical relative to the pattern of modifiednucleotides of the second strand.

The pattern of modified nucleotides of said first strand may be shiftedby one or more nucleotides relative to the pattern of modifiednucleotides of the second strand.

In some preferred embodiments the middle ribonucleotide in the firststrand (antisense) is an unmodified nucleotide. For example, in a19-oligomer antisense strand, ribonucleotide number 10 is unmodified; ina 21-oligomer antisense strand, ribonucleotide number 11 is unmodified;and in a 23-oligomer antisense strand, ribonucleotide number 12 isunmodified. The modifications or pattern of modification, if any, of thesiRNA must be planned to allow for this.

The modifications discussed above may be selected from the groupcomprising amino, fluoro, methoxy alkoxy, alkyl, amino, fluoro, chloro,bromo, CN, CF, imidazole, carboxylate, thioate, C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl, OCF₃, OCN, O-, S-, orN-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂, N₃;heterocycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino orsubstituted silyl, as, among others, described in European patents EP 0586 520 B1 or EP 0 618 925 B1.

The double stranded structure of the siRNA may be blunt ended, on one orboth sides. More specifically, the double stranded structure may beblunt ended on the double stranded structure's side which is defined bythe 5′-end of the first strand and the 3′-end of the second strand, orthe double stranded structure may be blunt ended on the double strandedstructure's side which is defined by at the 3′-end of the first strandand the 5′-end of the second strand.

Additionally, at least one of the two strands may have an overhang of atleast one nucleotide at the 5′-end; the overhang may consist of at leastone deoxyribonucleotide. At least one of the strands may also optionallyhave an overhang of at least one nucleotide at the 3′-end.

The length of the double-stranded structure of the siRNA is typicallyfrom about 17 to 21 and more preferably 18 or 19 bases. Further, thelength of said first strand and/or the length of said second strand mayindependently from each other be selected from the group comprising theranges of from about 15 to about 23 bases, 17 to 21 bases and 18 or 19bases. In another embodiment, the length of the double-strandedstructure of the siRNA is typically from about 17 to 23 and morepreferably 23 bases.

Additionally, the complementarily between said first strand and thetarget nucleic acid may be perfect, or the duplex formed between thefirst strand and the target nucleic acid may comprise at least 15nucleotides wherein there is one mismatch or two mismatches between saidfirst strand and the target nucleic acid forming said double-strandedstructure.

For example in a duplex region consisting of 19 base pairs one mismatchresults in 94.7% complementarity, two mismatches results in about 89.5%complementarity and 3 mismatches results in about 84.2% complementarity,rendering the duplex region substantially complementary. Accordinglysubstantially identical refers to identity of greater than about 84%, toanother sequence.

In some cases both the first strand and the second strand each compriseat least one group of modified nucleotides and at least one flankinggroup of nucleotides, whereby each group of modified nucleotidescomprises at least one nucleotide and whereby each flanking group ofnucleotides comprising at least one nucleotide with each group ofmodified nucleotides of the first strand being aligned with a flankinggroup of nucleotides on the second strand, whereby the most terminal 5′nucleotide of the first strand is a nucleotide of the group of modifiednucleotides, and the most terminal 3′ nucleotide of the second strand isa nucleotide of the flanking group of nucleotides. Each group ofmodified nucleotides may consist of a single nucleotide and/or eachflanking group of nucleotides may consist of a single nucleotide.

Additionally, it is possible that on the first strand the nucleotideforming the flanking group of nucleotides is an unmodified nucleotidewhich is arranged in a 3′ direction relative to the nucleotide formingthe group of modified nucleotides, and on the second strand thenucleotide forming the group of modified nucleotides is a modifiednucleotide which is arranged in 5′ direction relative to the nucleotideforming the flanking group of nucleotides.

Further the first strand of the siRNA may comprise eight to twelve,preferably nine to eleven, groups of modified nucleotides, and thesecond strand may comprise seven to eleven, preferably eight to ten,groups of modified nucleotides.

The first strand and the second strand may be linked by a loopstructure, which may be comprised of a non-nucleic acid polymer such as,inter alia, polyethylene glycol. Alternatively, the loop structure maybe comprised of a nucleic acid.

Further, the 5′-terminus of the first strand of the siRNA may be linkedto the 3′-terminus of the second strand, or the 3′-end of the firststrand may be linked to the 5′-terminus of the second strand, saidlinkage being via a nucleic acid linker typically having a lengthbetween 10-2000 nucleobases.

In one aspect the present invention provides a compound having thestructure:

5′ (N)_(x)-Z 3′ (antisense strand)

3′ Z′-(N′)_(y) 5′ (sense strand)

wherein each of N and N′ is a nucleotide which may be modified orunmodified in its sugar residue;

wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;

wherein each of x and y is an integer between 18 and 40;

wherein each of Z and Z′ may be present or absent, but if present is 1-5nucleotides covalently attached at the 3′ terminus of the strand inwhich it is present; and

wherein the sequence of (N)_(x) comprises an antisense sequence to mRNAof p53.

In some embodiments the compound comprises a phosphodiester bond. Invarious embodiments the compound comprises ribonucleotides wherein x=yand wherein x is an integer selected from the group consisting of 19,20, 21, 22 or 23. In some embodiments x=y=19. In other embodimentsx=y=23.

In some embodiments the compound is blunt ended, for example wherein Zand Z′ are both absent. In an alternative embodiment, the compoundcomprises at least one 3′ overhang, wherein at least one of Z or Z′ ispresent. Z and Z′ can be independently comprise one or more covalentlylinked modified or non-modified nucleotides, as described infra, forexample inverted dT or dA; dT, LNA, mirror nucleotide and the like. Insome embodiments each of Z and Z′ are independently selected from dT anddTdT.

In some embodiments the compound comprises one or more ribonucleotidesunmodified in their sugar residues. In other embodiments the compoundcomprises at least one ribonucleotide modified in the sugar residue. Insome embodiments the compound comprises a modification at the 2′position of the sugar residue. Modifications in the 2′ position of thesugar residue include amino, fluoro, methoxy, alkoxy and alkyl moieties.In certain preferred embodiments the modification comprises aribonucleotide comprising a methoxy moiety at the 2′ position(2′-O-methyl; 2′-O-Me; 2′-O—CH₃) of the sugar residue.

In some embodiments the compound comprises modified alternatingribonucleotides in one or both of the antisense and the sense strands.In preferred embodiments the compound comprises modified alternatingribonucleotides in the antisense and the sense strands. In somepreferred embodiments the middle ribonucleotide of the antisense strandis not modified; e.g. ribonucleotide in position 10 in a 19-mer strand.

In additional embodiments the compound comprises modifiedribonucleotides in alternating positions wherein the ribonucleotides atthe 5′ and 3′ termini of the antisense strand are modified in theirsugar residues, and the ribonucleotides at the 5′ and 3′ termini of thesense strand are unmodified in their sugar residues. In someembodiments, neither the antisense nor the sense strands arephosphorylated at the 3′ and 5′ termini. In other embodiments either orboth the antisense and the sense strands are phosphorylated at the 3′termini.

In various embodiments the compound comprises an antisense sequencepresent in Tables A, B, C or C2. In other embodiments the presentinvention provides a mammalian expression vector comprising an antisensesequence present in Tables A, B, C or C2.

In certain embodiments the present invention provides a compound havingthe structure

-   -   5′ (N) 3′ antisense strand    -   3′ (N′) 5′ sense strand

wherein each of N and N′=19 and are fully complementary;

wherein alternating ribonucleotides in the antisense and the sensestrands are modified to result in a 2′-O-methyl modification in thesugar residue of the ribonucleotides;

wherein the ribonucleotides at the 5′ and 3′ termini of the antisensestrand are modified;

wherein the ribonucleotides at the 5′ and 3′ termini of the sense strandare unmodified;

wherein the antisense and the sense strands are phosphorylated ornon-phosphorylated at the 3′ and 5′ termini; and

wherein each of N and N′ is selected from the group of oligomers setforth in Tables A, B, or C.

In certain embodiments the present invention provides a compound havingthe structure

-   -   5′ (N)x 3′ antisense strand    -   3′ (N′)y 5′ sense strand

wherein each of x and y=23 and (N)_(x) and (N′)_(y) are fullycomplementary

wherein alternating ribonucleotides in (N)_(x) and (N′)_(y) are modifiedto result in a 2′-O-methyl modification in the sugar residue of theribonucleotides;

wherein each N at the 5′ and 3′ termini of (N)_(x) are modified;

wherein each N′ at the 5′ and 3′ termini of (N′)_(y) are unmodified;

wherein each of (N)_(x) and (N′)_(y) is selected from the group ofoligomers set forth in Table C2.

(N)_(x) and (N′)_(y) may be phosphorylated or non-phosphorylated at the3′ and 5′ termini. In certain embodiments of the invention, alternatingribonucleotides are modified in both the antisense and the sense strandsof the compound. In particular the exemplified siRNA has been modifiedsuch that a 2′-O-methyl (2′-OMe) group was present on the first, third,fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth,nineteenth, twenty-first and twenty-third nucleotide of the antisensestrand (N)_(x), and whereby the very same modification, i.e. a 2′-OMegroup, was present at the second, fourth, sixth, eighth, tenth, twelfth,fourteenth, sixteenth, eighteenth, twentieth and twenty-secondnucleotide of the sense strand (N′)_(y). Additionally, it is to be notedthat these particular siRNA compounds are also blunt ended.

In certain embodiments of the compounds of the invention havingalternating ribonucleotides modified in one or both of the antisense andthe sense strands of the compound; for 19-mers and 23-mers theribonucleotides at the 5′ and 3′ termini of the antisense strand aremodified in their sugar residues, and the ribonucleotides at the 5′ and3′ termini of the sense strand are unmodified in their sugar residues.For 21-mers the ribonucleotides at the 5′ and 3′ termini of the sensestrand are modified in their sugar residues, and the ribonucleotides atthe 5′ and 3′ termini of the antisense strand are unmodified in theirsugar residues. As mentioned above, it is preferred that the middlenucleotide of the antisense strand is unmodified.

In preferred embodiments of the invention, alternating ribonucleotidesare modified in both the antisense and the sense strands of thecompound. In particular the siRNA used in the Examples has been suchmodified such that a 2′ O-Me group was present on the first, third,fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth andnineteenth nucleotide of the antisense strand, whereby the very samemodification, i.e. a 2′-O-Me group was present at the second, fourth,sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenthnucleotide of the sense strand. Additionally, it is to be noted that thein case of these particular nucleic acids according to the presentinvention the first stretch is identical to the first strand and thesecond stretch is identical to the second strand and these nucleic acidsare also blunt ended.

In a particularly preferred embodiment the sequence of the siRNA is thatof 15 in Table A (SEQ ID NO 25).

According to one preferred embodiment of the invention, the antisenseand the sense strands of the siRNA molecule are both phosphorylated onlyat the 3′-terminus and not at the 5′-terminus. According to anotherpreferred embodiment of the invention, the antisense and the sensestrands are both non-phosphorylated both at the 3′-terminus and also atthe 5′-terminus.

According to yet another preferred embodiment of the invention, the1^(st) nucleotide in the 5′ position in the sense strand is specificallymodified to abolish any possibility of in vivo 5′-phosphorylation.

In various preferred embodiments of the compounds of the inventionhaving alternating ribonucleotides modified in both the antisense andthe sense strands of the compound, for 19-mer oligomers and 23-meroligomers the ribonucleotides at the 5′ and 3′ termini of the antisensestrand are modified in their sugar residues, and the ribonucleotides atthe 5′ and 3′ termini of the sense strand are unmodified in their sugarresidues. For 21-mer oligomers the ribonucleotides at the 5′ and 3′termini of the sense strand are modified in their sugar residues, andthe ribonucleotides at the 5′ and 3′ termini of the antisense strand areunmodified in their sugar residues. As mentioned above, it is preferredthat the middle nucleotide of the antisense strand is unmodified.

The invention further provides a vector capable of expressing any of theaforementioned oligoribonucleotides in unmodified form in a cell afterwhich appropriate modification may be made.

The invention also provides a composition comprising one or more of thecompounds of the invention in a carrier, preferably a pharmaceuticallyacceptable carrier. This composition may comprise a mixture of two ormore different siRNAs.

The invention also provides a composition which comprises the abovecompound of the invention covalently or non-covalently bound to one ormore compounds of the invention in an amount effective to inhibit humanp53 and a carrier. This composition may be processed intracellularly byendogenous cellular complexes to produce one or moreoligoribonucleotides of the invention.

The invention also provides a composition comprising a carrier and oneor more of the compounds of the invention in an amount effective todown-regulate expression in a cell of a human p53, which compoundcomprises a sequence substantially complementary to the sequence of(N)_(x).

Additionally the invention provides a method of down-regulating theexpression of gene p53 by at least 50% as compared to a controlcomprising contacting an mRNA transcript of gene p53 with one or more ofthe compounds of the invention.

Additionally, the invention provides a method of inhibiting theexpression of p53 by at least 20%, preferably 30%, even more preferably40% or even 50% as compared to a control comprising contacting an mRNAtranscript of p53 with one or more of the compounds of the invention.

In one embodiment the oligoribonucleotide is down-regulating p53,whereby the down-regulation of p53 is selected from the group comprisingdown-regulation of gene function, down-regulation of polypeptide anddown-regulation of mRNA expression.

In one embodiment the compound is down-regulating a p53 polypeptide,whereby the down-regulation is selected from the group comprisingdown-regulation of function (which may be examined by an enzymatic assayor a binding assay with a known interactor of the nativegene/polypeptide, inter alia), down-regulation of protein (which may beexamined by Western blotting, ELISA or immuno-precipitation, inter alia)and down-regulation of mRNA expression (which may be examined byNorthern blotting, quantitative RT-PCR, in-situ hybridisation ormicroarray hybridisation, inter alia).

More particularly, the invention provides an oligoribonucleotide whereinone strand comprises consecutive nucleotides having, from 5′ to 3′, thesequence set forth in SEQ ID NOS: 3-25 (Table A, sense strands) or inSEQ ID NOS: 49-119 (Table B, sense strands) or in SEQ ID NOS: 191-253(Table C, sense strands), or in SEQ ID NOS: 317-352 (Table C2, sensestrands) or a homolog thereof wherein in up to 2 of the nucleotides ineach terminal region a base is altered.

The terminal region of the oligonucleotide refers to bases 1-4 and/or16-19 in the 19-mer sequence, or to bases 1-4 and/or 18-21 in the 21-mersequence or to bases 1-4 and/or 20-23 in the 23-mer sequence.

Additionally, the invention provides oligoribonucleotides wherein onestrand comprises consecutive nucleotides having, from 5′ to 3′, thesequence set forth SEQ ID NOS: 26-48 (Table A, antisense strands) or SEQID NOS: 120-190 (Table B, antisense strands) or SEQ ID NOS: 254-316(Table C, antisense strands), or SEQ ID NOS: 353-387 (Table C2,antisense strands) or a homolog thereof wherein in up to 2 of thenucleotides in each terminal region a base is altered.

Preferred list of siRNA (sense and antisense strands) directed to p53are in Tables A, B, C and C2.

The preferred oligonucleotides of the invention are human p53oligonucleotides serial numbers 3, 5, 20 and 23 in Table D and mouse p53oligonucleotides serial numbers 1 11, 12, 14, 17 and 18 in Table E.These are identical to serial numbers 3, 5, 20 and 23 (human) and also11, 12, 14, 17 and 18 (mouse) in Table A. The most preferredoligonucleotides of the invention are human p53 oligonucleotides havingthe sequence of serial number 23 in Table A.

The presently most preferred compound of the invention is a blunt-ended19-mer oligonucleotide, i.e. x=y=19 and Z and Z′ are both absent. Theoligonucleotide molecule is either phosphorylated at 3′ termini of bothsense and anti-sense strands, or non-phosphorylated at all; or having1^(st) nucleotide in the 5′ position on the sense strand specificallymodified to abolish any possibility of in vivo 5′-phosphorylation. Thealternating ribonucleotides are modified at the 2′ position in both theantisense and the sense strands, wherein the moiety at the 2′ positionis methoxy (2′-O-methyl) and wherein the ribonucleotides at the 5′ and3′ termini of the antisense strand are modified in their sugar residues,and the ribonucleotides at the 5′ and 3′ termini of the sense strand areunmodified in their sugar residues. The presently most preferred suchcompounds are such modified oligonucleotides comprising the sequenceshaving serial number 23 in Table A.

In one aspect of the invention the oligonucleotide comprises adouble-stranded structure, whereby such double-stranded structurecomprises

-   -   a first strand and a second strand, whereby    -   the first strand comprises a first stretch of contiguous        nucleotides and the second strand comprises a second stretch of        contiguous nucleotides, whereby    -   the first stretch is either complementary or identical to a        nucleic acid sequence coding for p53 and whereby the second        stretch is either identical or complementary to a nucleic acid        sequence coding for p53.

In an embodiment the first stretch and/or the second stretch comprisesfrom about 14 to 40 nucleotides, preferably about 18 to 30 nucleotides,more preferably from about 19 to 27 nucleotides and most preferably fromabout 19 to 23 nucleotides, in particular from about 19 to 21 or fromabout 19 to 23 nucleotides. In such an embodiment the oligonucleotidemay be from 17-40 nucleotides in length.

Additionally, further nucleic acids according to the present inventioncomprise at least 14 contiguous nucleotides of any one of thepolynucleotides in Tables A, B, C or C2 and more preferably 14contiguous nucleotide base pairs at any end of the double-strandedstructure comprised of the first stretch and second stretch as describedabove.

In an embodiment the first stretch comprises a sequence of at least 14contiguous nucleotides of an oligonucleotide, whereby sucholigonucleotide is selected from the group comprising SEQ. ID. NOS3-387, preferably from the group comprising the oligoribonucleotides ofhaving the sequence of any of the serial numbers 3, 5, 20 or 23 (human)or having the sequence of any of the serial numbers 11, 12, 14, 17 and18 (mouse) in Table A, more preferably selected from the group havingthe sequence of any of the serial numbers 3, 5, 20 or 23 in Table A.

Additionally, further nucleic acids according to the present inventioncomprise at least 14 contiguous nucleotides of any one of the SEQ. ID.NO. 3 to 387, and more preferably 14 contiguous nucleotide base pairs atany end of the double-stranded structure comprised of the first stretchand second stretch as described above. It will be understood by oneskilled in the art that given the potential length of the nucleic acidaccording to the present invention and particularly of the individualstretches forming such nucleic acid according to the present invention,some shifts relative to the coding sequence of p53 to each side ispossible, whereby such shifts can be up to 1, 2, 3, 4, 5 and 6nucleotides in both directions, and whereby the thus generateddouble-stranded nucleic acid molecules shall also be within the presentinvention.

In another aspect, the present invention relates to a method for thetreatment of a subject in need of treatment for a disease or disorderassociated with the abnormal expression of p53, comprising administeringto the subject an amount of an inhibitor which reduces or inhibitsexpression of p53.

The methods of the invention comprise administering to the patient oneor more inhibitory compounds which down-regulate expression of p53; andin particular siRNA in a therapeutically effective dose so as to therebytreat the patient.

In various embodiments the inhibitor is selected from the groupconsisting of an siRNA, shRNA, an aptamer, an antisense molecule, miRNA,a ribozyme, and an antibody. In preferred embodiments the inhibitor issiRNA.

Delivery: The siRNA molecules of the present invention may be deliveredto the target tissue (such as the cochlea) by direct application of thenaked molecules admixed with a carrier or a diluent within the cochlea.

The term “naked siRNA” refers to siRNA molecules that are free from anydelivery vehicle that acts to assist, promote or facilitate entry intothe cell, including viral sequences, viral particles, liposomeformulations, lipofectin or precipitating agents and the like. Forexample, siRNA in PBS is “naked siRNA”. However, the siRNA molecules ofthe invention can also be delivered in liposome formulations andlipofectin formulations and the like and can be prepared by methods wellknown to those skilled in the art. Such methods are described, forexample, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, whichare herein incorporated by reference.

Delivery systems aimed specifically at the enhanced and improveddelivery of siRNA into mammalian cells have been developed, see, forexample, Shen et al (FEBS letters 539: 111-114 (2003)), Xia et al.,Nature Biotechnology 20: 1006-1010 (2002), Reich et al., MolecularVision 9: 210-216 (2003), Sorensen et al. (J. Mol. Biol. 327: 761-766(2003), Lewis et al., Nature Genetics 32: 107-108 (2002) and Simeoni etal., Nucleic Acids Research 31, 11: 2717-2724 (2003). siRNA has recentlybeen successfully used for inhibition in primates; for further detailssee Tolentino et al., Retina 24(1) February 2004 I 132-138. Respiratoryformulations for siRNA are described in U.S. patent application No.2004/0063654 of Davis et al. Cholesterol-conjugated siRNAs (and othersteroid and lipid conjugated siRNAs) can been used for delivery seeSoutschek et al Nature 432: 173-177(2004) Therapeutic silencing of anendogenous gene by systemic administration of modified siRNAs; andLorenz et al. Bioorg. Med. Chemistry. Lett. 14:4975-4977 (2004) Steroidand lipid conjugates of siRNAs to enhance cellular uptake and genesilencing in liver cells.

The siRNAs or pharmaceutical compositions of the present invention areadministered and dosed in accordance with good medical practice, takinginto account the clinical condition of the individual patient, thedisease to be treated, the site and method of administration, schedulingof administration, patient age, sex, body weight and other factors knownto medical practitioners.

The “therapeutically effective dose” for purposes herein is thusdetermined by such considerations as are known in the art. The dose mustbe effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art. The compounds of thepresent invention can be administered by any of the conventional routesof administration. It should be noted that the compound can beadministered as the compound or as pharmaceutically acceptable salt andcan be administered alone or as an active ingredient in combination withpharmaceutically acceptable carriers, solvents, diluents, excipients,adjuvants and vehicles. The compounds can be administered orally,subcutaneously or parenterally including intravenous, intraarterial,intramuscular, intraperitoneally, and intranasal administration as wellas intrathecal and infusion techniques. Implants of the compounds arealso useful. Liquid forms may be prepared for injection, the termincluding subcutaneous, transdermal, intravenous, intramuscular,intrathecal, and other parental routes of administration. The liquidcompositions include aqueous solutions, with and without organicco-solvents, aqueous or oil suspensions, emulsions with edible oils, aswell as similar pharmaceutical vehicles. In addition, under certaincircumstances the compositions for use in the novel treatments of thepresent invention may be formed as aerosols, for intranasal and likeadministration. The patient being treated is a warm-blooded animal and,in particular, mammals including man. The pharmaceutically acceptablecarriers, solvents, diluents, excipients, adjuvants and vehicles as wellas implant carriers generally refer to inert, non-toxic solid or liquidfillers, diluents or encapsulating material not reacting with the activeingredients of the invention and they include liposomes andmicrospheres. Examples of delivery systems useful in the presentinvention include U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;4,439,196; and 4,475,196. Many other such implants, delivery systems,and modules are well known to those skilled in the art. In one specificembodiment of this invention topical and transdermal formulations areparticularly preferred.

In general, the active dose of compound for humans is in the range offrom 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of onedose per day or twice or three or more times per day for a period of 1-4weeks or longer. In a particular embodiment, the administrationcomprises intravenous administration of the siRNA compound. Preferreddoses are in the range of 0.1-25 mg/kg body weight, more preferably, inthe range of 0.5-10 mg/kg body weight.

In another particular embodiment the administration comprises topical orlocal administration

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) pro-apoptotic-related disorder as listed above. Those in needof treatment include those already experiencing the disease orcondition, those prone to having the disease or condition, and those inwhich the disease or condition is to be prevented. The compounds of theinvention may be administered before, during or subsequent to the onsetof the disease or condition.

In another aspect of the invention a pharmaceutical composition isprovided which comprises any of the above oligoribonucleotides (SEQ IDNOS: 3-387) or vectors and a pharmaceutically acceptable carrier.Another aspect of the invention is the use of a therapeuticallyeffective amount of any of the above oligoribonucleotides (SEQ ID NOS:3-387) or vectors for the preparation of a medicament for treating apatient suffering from a disorder which is accompanied by an elevatedlevel of p53.

The present invention relates to the use of compounds whichdown-regulate the expression of the p53 gene, particularly to smallinterfering RNAs (siRNAs), in the treatment of hearing impairment oracute renal failure. Methods, molecules and compositions which inhibitp53 are discussed herein at length, and any of said molecules and/orcompositions may be beneficially employed in the treatment of a patientsuffering from any of said conditions. Preferred lists of siRNA directedto p53 are in Tables A, B, C and C2. Other siRNA sequences directed toRTP801 to be used in the present invention may be found in co-pendingPCT publication number WO06/023544A2 (PCT/US2005/029236) or U.S.application Ser. No. 11/207,119, which are incorporated by reference intheir entirety.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) an apoptotic-related disorder such as hearing disorder orimpairment (or balance impairment), preferably ototoxin-induced ortraumatic inner ear hair cells apoptotic damage. Those in need oftreatment include those already experiencing a hearing impairment, thoseprone to having the impairment, and those in which the impairment is tobe prevented. Without being bound by theory, the hearing impairment maybe due to apoptotic inner ear hair cell damage or loss, wherein thedamage or loss is caused by infection, mechanical injury, loud sound,aging, or, in particular, chemical-induced ototoxicity. Ototoxinsinclude therapeutic drugs including antineoplastic agents, salicylates,quinines, and aminoglycoside antibiotics, contaminants in foods ormedicinals, and environmental or industrial pollutants. Typically,treatment is performed to prevent or reduce ototoxicity, especiallyresulting from or expected to result from administration of therapeuticdrugs. Preferably a therapeutically effective composition is givenimmediately after the exposure to prevent or reduce the ototoxic effect.More preferably, treatment is provided prophylactically, either byadministration of the composition prior to or concomitantly with theototoxic pharmaceutical or the exposure to the ototoxin.

By “ototoxin” in the context of the present invention is meant asubstance that through its chemical action injures, impairs or inhibitsthe activity of the sound receptors component of the nervous systemrelated to hearing, which in turn impairs hearing (and/or balance). Inthe context of the present invention, ototoxicity includes a deleteriouseffect on the inner ear hair cells. Ototoxic agents that cause hearingimpairments include, but are not limited to, neoplastic agents such asvincristine, vinblastine, cisplatin and cisplatin-like compounds, taxoland taxol-like compounds, dideoxy-compounds, e.g., dideoxyinosine;alcohol; metals; industrial toxins involved in occupational orenvironmental exposure; contaminants of food or medicinals; andover-doses of vitamins or therapeutic drugs, e.g., antibiotics such aspenicillin or chloramphenicol, and megadoses of vitamins A, D, or B6,salicylates, quinines and loop diuretics. By “exposure to an ototoxicagent” is meant that the ototoxic agent is made available to, or comesinto contact with, a mammal. Exposure to an ototoxic agent can occur bydirect administration, e.g., by ingestion or administration of a food,medicinal, or therapeutic agent, e.g., a chemotherapeutic agent, byaccidental contamination, or by environmental exposure, e.g., aerial oraqueous exposure.

Hearing impairment relevant to the invention may be due to end-organlesions involving inner ear hair cells, e.g., acoustic trauma, viralendolymphatic labyrinthitis, Meniere's disease. Hearing impairmentsinclude tinnitus, which is a perception of sound in the absence of anacoustic stimulus, and may be intermittent or continuous, wherein thereis diagnosed a sensorineural loss. Hearing loss may be due to bacterialor viral infection, such as in herpes zoster oticus, purulentlabyrinthitis arising from acute otitis media, purulent meningitis,chronic otitis media, sudden deafness including that of viral origin,e.g., viral endolymphatic labyrinthitis caused by viruses includingmumps, measles, influenza, chicken pox, mononucleosis and adenoviruses.The hearing loss can be congenital, such as that caused by rubella,anoxia during birth, bleeding into the inner ear due to trauma duringdelivery, ototoxic drugs administered to the mother, erythroblastosisfetalis, and hereditary conditions including Waardenburg's syndrome andHurler's syndrome.

The hearing loss can be noise-induced, generally due to a noise greaterthan 85 decibels (db) that damages the inner ear. In a particular aspectof the invention, the hearing loss is caused by an ototoxic drug thateffects the auditory portion of the inner ear, particularly inner earhair cells. Incorporated herein by reference are chapters 196, 197, 198and 199 of The Merck Manual of Diagnosis and Therapy, 14th Edition,(1982), Merck Sharp & Dome Research Laboratories, N.J. and correspondingchapters in the most recent 16th edition, including Chapters 207 and210) relating to description and diagnosis of hearing and balanceimpairments.

In one embodiment, the present invention constitutes a method fortreating a mammal having or prone to a hearing (or balance) impairmentor treating a mammal prophylactically in conditions where inhibition ofexpression of p53 is beneficial. The method of the present inventionwould prevent or reduce the occurrence or severity of a hearing (orbalance) impairment that would result from inner ear cell injury, loss,or degeneration, in particular caused by an ototoxic agent. The methodof the invention includes administering a therapeutically effectiveamount of one or more compounds which down-regulate expression of thep53 gene, particularly the novel siRNAs of the present invention, smallmolecule inhibitors of p53 as described herein or antibodies to p53polypeptide.

It is the object of the present invention to provide a method andcompositions for treating a mammal, to prevent, reduce, or treat ahearing impairment, disorder or imbalance, preferably anototoxin-induced hearing condition, by administering to a mammal in needof such treatment a composition of the invention. One embodiment of theinvention is a method for treating a hearing disorder or impairmentwherein the ototoxicity results from administration of a therapeuticallyeffective amount of an ototoxic pharmaceutical drug. Typical ototoxicdrugs are chemotherapeutic agents, e.g. antineoplastic agents, andantibiotics. Other possible candidates include loop-diuretics, quininesor a quinine-like compound, and salicylate or salicylate-like compounds.

The methods and compositions of the present invention are also effectivewhen the ototoxic compound is an antibiotic, preferably anaminoglycoside antibiotic. Ototoxic aminoglycoside antibiotics includebut are not limited to neomycin, paromomycin, ribostamycin, lividomycin,kanamycin, amikacin, tobramycin, viomycin, gentamicin, sisomicin,netilmicin, streptomycin, dibekacin, fortimicin, anddihydrostreptomycin, or combinations thereof. Particular antibioticsinclude neomycin B, kanamycin A, kanamycin B, gentamicin C1, gentamicinC1a, and gentamicin C2.

The methods and compositions of the present invention are also effectivewhen the ototoxic compound is a neoplastic agent such as vincristine,vinblastine, cisplatin and cisplatin-like compounds and taxol andtaxol-like compounds.

The methods and compositions of the present invention are also effectivein the treatment of acoustic trauma or mechanical trauma, preferablyacoustic or mechanical trauma that leads to inner ear hair cell loss orouter ear hair cell loss. Acoustic trauma to be treated in the presentinvention may be caused by a single exposure to an extremely loud soundof above 120-140 decibels, or following long-term exposure to everydayloud sounds above 85 decibels. The compositions of the present inventionare also effective as a preventive treatment in patients expecting anacoustic trauma. Mechanical inner ear trauma to be treated in thepresent invention is for example the inner ear trauma following anoperation for insertion of an electronic device in the inner ear. Thecompositions of the present invention prevent or minimize the damage toinner ear hair cells associated with this operation.

In some embodiments the composition of the invention is co-administeredwith an ototoxin. For example, an improved method is provided fortreatment of infection of a mammal by administration of anaminoglycoside antibiotic, the improvement comprising administering atherapeutically effective amount of one or more compounds (particularlynovel siRNAs) which down-regulate expression of the p53 gene, to thepatient in need of such treatment to reduce or prevent ototoxin-inducedhearing impairment associated with the antibiotic. The compounds whichdown-regulate expression of the p53 gene, particularly novel siRNAs arepreferably administered locally within the inner ear.

In yet another embodiment an improved method for treatment of cancer ina mammal by administration of a chemotherapeutic compound is provided,wherein the improvement comprises administering a therapeuticallyeffective amount of a composition of the invention to the patient inneed of such treatment to reduce or prevent ototoxin-induced hearingimpairment associated with the chemotherapeutic drug. The compoundswhich reduce or prevent the ototoxin-induced hearing impairment, eg thenovel siRNAs inter alia are preferable administered directly to thecochlea as naked siRNA in a vehicle such as PBS or other physiologicalsolutions, but may alternatively be administered with a delivery vehicleas described above.

In another embodiment the methods of treatment are applied to treatmentof hearing impairment resulting from the administration of achemotherapeutic agent in order to treat its ototoxic side-effect.Ototoxic chemotherapeutic agents amenable to the methods of theinvention include, but are not limited to an antineoplastic agent,including cisplatin or cisplatin-like compounds, taxol or taxol-likecompounds, and other chemotherapeutic agents believed to causeototoxin-induced hearing impairments, e.g., vincristine, anantineoplastic drug used to treat hematological malignancies andsarcomas. Cisplatin-like compounds include carboplatin (Paraplatin®),tetraplatin, oxaliplatin, aroplatin and transplatin inter alia

In another embodiment the methods of the invention are applied tohearing impairments resulting from the administration of quinine and itssynthetic substitutes, typically used in the treatment of malaria, totreat its ototoxic side-effect.

In another embodiment the methods of the invention are applied tohearing impairments resulting from administration of a diuretic to treatits ototoxic side-effect. Diuretics, particularly “loop” diuretics, i.e.those that act primarily in the Loop of Henle, are candidate ototoxins.Illustrative examples, not limiting to the invention method, includefurosemide, ethacrylic acid, and mercurials. Diuretics are typicallyused to prevent or eliminate edema. Diuretics are also used innonedematous states for example hypertension, hypercalcemia, idiopathichypercalciuria, and nephrogenic diabetes insipidus.

In another preferred embodiment, the compositions of the presentinvention are used for the treatment of acute renal failure. In yetanother preferred embodiment, the compositions of the present inventionare used in conditions in which a p53 gene is activated as a consequenceof a variety of stresses associated with injuries such as a burn,hyperthermia, hypoxia associated with a blocked blood supply such as inmyocardial infraction, stroke, and ischemia. Temporary p53 inhibitionusing the siRNA molecules of the present invention can betherapeutically effective in reducing or eliminating p53-dependentneuronal death in the central nervous system, i.e., brain and spinalcord injury, in preserving of tissue and an organ prior totransplanting, preparation of a host for a bone marrow transplant,reducing or eliminating neuronal damage during a seizure and insuppressing tissue aging.

The invention also provides a use of a therapeutically effective dose ofone or more compounds of the invention for the preparation of acomposition for the treatment of a disease accompanied by an elevatedlevel of p53, such as in a patient suffering from acute kidney injurysuch as acute renal failure. Preferably, the composition is for thetreatment of renal ischemia-reperfusion injury, most preferably renalischemia-reperfusion injury in patients undergoing major surgery such asmajor cardiac surgery which may result in acute renal failure.

In a particular embodiment in which the siRNA compounds are delivered tothe target cells in the kidney, the siRNA compounds are preferablyadministered by intravenous injection. The inventors have unexpectedlydiscovered that a single injection of p53 siRNA compounds between 2 to24, preferably 2-8 hours following the kidney ischemic-reperfusion eventin animals is especially effective in reducing the damage in the kidney.In the most preferred embodiment, the p53 siRNA compounds are injected 4hours following the kidney ischemic-reperfusion event or following theremoval of the cardiopulmonary bypass machine. It is also envisaged thattwo or more injections may be administered in this time period andinjections may be given 1, 2, 3, 4, 5, 6, 7, 8, 12 or 24 hours followingthe kidney ischemic-reperfusion event or following the removal of thecardiopulmonary bypass machine

It is noted that the delivery of the siRNA compounds according to thepresent invention to the target cells in the kidney proximal tubules isparticularity effective in the treatment of acute renal failure. Withoutbeing bound by theory, this may be due to the fact that siRNA moleculesare excreted from the body via the cells of the kidney proximal tubules.Thus, naked siRNA molecules are naturally concentrated in the cells thatare targeted for the therapy in acute renal failure.

The compounds of the invention are preferably used for treating acuterenal failure, in particular acute renal failure due to ischemia in postsurgical patients, and acute renal failure due to chemotherapy treatmentsuch as cisplatin administration or sepsis-associated acute renalfailure. A preferred use of the compounds of the invention is for theprevention of acute renal failure in high-risk patients undergoing majorcardiac surgery or vascular surgery. The patients at high-risk ofdeveloping acute renal failure can be identified using various scoringmethods such as the Cleveland Clinic algorithm or that developed by USAcademic Hospitals (QMMI) and by Veterans' Administration (CICSS).

Other preferred uses of the compounds of the invention are for theprevention of ischemic acute renal failure in kidney transplant patientsor for the prevention of toxic acute renal failure in patients receivingchemotherapy or other chemicals causing nephrotoxicity. Suchnephrotoxins are for example: cardiovascular drugs such as diuretics,β-blockers, vasodilator agents, ACE inhibitors, ciclosporin,Aminoglycoside antibiotics (e.g. gentamicin), amphotericin B, cisplatin,radiocontrast media, immunoglobulins, mannitol, NSAIDs (e.g. aspirin,ibuprofen, diclofenac), ciclosporin, lithium salts, cyclophosphamide,amphotericin B, sulphonamides, methotrexate, aciclovir, polyethyleneglycol, β-lactam antibiotics, vancomycin, rifampicin, sulphonamides,ciprofloxacin, ranitidine, cimetidine, furosemide, thiazides, phenyloin,Penicillamine, amphotericin B, fluoride, demeclocycline, foscarnet,Heavy metals.

Other uses are for wound healing, acute liver failure, drug-induceddeafness (perhaps topically), ex vivo expansion of hematopoietic stemcells, preservation of donor organs/tissues before transplantation bysoaking in siRNA solution (perhaps by electroporation) and subsequentimprovement of graft tissue survival following transplantation. Otherindications may be stroke, Parkinson's disease, Alzheimer's disease,doxorubicin-induced cardiotoxicity, myocardial infarction/heart failureand improvement of graft tissue survival following transplantation (bysystemic administration). Without being bound by theory all thesedisorders are accompanied by an elevated level of p53 polypeptide.

The present invention also provides for a process of preparing apharmaceutical composition, which comprises:

-   -   obtaining one or more double stranded compound of the invention;        and    -   admixing said compound with a pharmaceutically acceptable        carrier.

The present invention also provides for a process of preparing apharmaceutical composition, which comprises admixing one or morecompounds of the present invention with a pharmaceutically acceptablecarrier.

In a preferred embodiment, the compound used in the preparation of apharmaceutical composition is admixed with a carrier in apharmaceutically effective dose. In a particular embodiment the compoundof the present invention is conjugated to a steroid or to a lipid or toanother suitable molecule e.g. to cholesterol.

Modifications or analogs of nucleotides can be introduced to improve thetherapeutic properties of the nucleotides. Improved properties includeincreased nuclease resistance and/or increased ability to permeate cellmembranes.

Accordingly, the present invention also includes all analogs of, ormodifications to, a oligonucleotide of the invention that does notsubstantially affect the function of the polynucleotide oroligonucleotide. In a preferred embodiment such modification is relatedto the base moiety of the nucleotide, to the sugar moiety of thenucleotide and/or to the phosphate moiety of the nucleotide.

In embodiments of the invention, the nucleotides can be selected fromnaturally occurring or synthetically modified bases. Naturally occurringbases include adenine, guanine, cytosine, thymine and uracil. Modifiedbases of the oligonucleotides include inosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl-adenines, 5-halouracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudouracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine,8-thioalkyl adenine, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanine, 8-amino guanine, 8-thiol guanine, 8-thioalkylguanine, 8-hydroxyl guanine and other substituted guanines, other azaand deaza adenines, other aza and deaza guanines, 5-trifluoromethyluracil and 5-trifluoro cytosine.

In addition, analogs of polynucleotides can be prepared wherein thestructure of one or more nucleotide is fundamentally altered and bettersuited as therapeutic or experimental reagents. An example of anucleotide analogue is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA is replacedwith a polyamide backbone which is similar to that found in peptides.PNA analogues have been shown to be resistant to enzymatic degradationand to have extended stability in vivo and in vitro. Other modificationsthat can be made to oligonucleotides include polymer backbones, cyclicbackbones, acyclic backbones, thiophosphate-D-ribose backbones, triesterbackbones, thioate backbones, 2′-5′ bridged backbone, artificial nucleicacids, morpholino nucleic acids, locked nucleic acid (LNA), glycolnucleic acid (GNA), threose nucleic acid (TNA), arabinoside, and mirrornucleoside (for example, beta-L-deoxynucleoside instead ofbeta-D-deoxynucleoside). Examples of siRNA compounds comprising LNAnucleotides are disclosed in Elmen et al., (NAR 2005. 33(1):439-447).

The compounds of the present invention can be synthesized using one ormore inverted nucleotides, for example inverted thymidine or invertedadenine (for example see Takei, et al., 2002. JBC 277(26):23800-06.

Certain structures include an siRNA compound having one or a pluralityof 2′-5′ internucleotide linkages (bridges).

A “mirror” nucleotide is a nucleotide with reversed chirality to thenaturally occurring or commonly employed nucleotide, i.e., a mirrorimage (L-nucleotide) of the naturally occurring (D-nucleotide). Thenucleotide can be a ribonucleotide or a deoxyribonucleotide and myfurther comprise at least one sugar, base and or backbone modification.U.S. Pat. No. 6,602,858 discloses nucleic acid catalysts comprising atleast one L-nucleotide substitution.

In one embodiment the modification is a modification of the phosphatemoiety, whereby the modified phosphate moiety is selected from the groupcomprising phosphothioate.

The compounds of the present invention can be synthesized by any of themethods that are well-known in the art for synthesis of ribonucleic (ordeoxyribonucleic) oligonucleotides. Such synthesis is, among others,described in Beaucage S. L. and Iyer R. P., Tetrahedron 1992; 48:2223-2311, Beaucage S. L. and Iyer R. P., Tetrahedron 1993; 49:6123-6194 and Caruthers M. H. et. al., Methods Enzymol. 1987; 154:287-313; the synthesis of thioates is, among others, described inEckstein F., Annu. Rev. Biochem. 1985; 54: 367-402, the synthesis of RNAmolecules is described in Sproat B., in Humana Press 2005 edited byHerdewijn P.; Kap. 2: 17-31 and respective downstream processes are,among others, described in Pingoud A. et. al., in IRL Press 1989 editedby Oliver R. W. A.; Kap. 7: 183-208 and Sproat B., in Humana Press 2005edited by Herdewijn P.; Kap. 2: 17-31 (supra).

Other synthetic procedures are known in the art e.g. the procedures asdescribed in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringeet al., 1990, Nucleic Acids Res., 18, 5433; Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684; and Wincott et al., 1997, Methods Mol.Bio., 74, 59, and these procedures may make use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end. The modified (e.g. 2′-O-methylated)nucleotides and unmodified nucleotides are incorporated as desired.

The oligonucleotides of the present invention can be synthesizedseparately and joined together post-synthetically, for example, byligation (Moore et al., 1992, Science 256, 9923; Draper et al.,International PCT publication No. WO93/23569; Shabarova et al., 1991,Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides &Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

It is noted that a commercially available machine (available, interalia, from Applied Biosystems) can be used; the oligonucleotides areprepared according to the sequences disclosed herein. Overlapping pairsof chemically synthesized fragments can be ligated using methods wellknown in the art (e.g., see U.S. Pat. No. 6,121,426). The strands aresynthesized separately and then are annealed to each other in the tube.Then, the double-stranded siRNAs are separated from the single-strandedoligonucleotides that were not annealed (e.g. because of the excess ofone of them) by HPLC. In relation to the siRNAs or siRNA fragments ofthe present invention, two or more such sequences can be synthesized andlinked together for use in the present invention.

The compounds of the invention can also be synthesized via a tandemsynthesis methodology, as described in US patent application publicationNo. US2004/0019001 (McSwiggen), wherein both siRNA strands aresynthesized as a single contiguous oligonucleotide fragment or strandseparated by a cleavable linker which is subsequently cleaved to provideseparate siRNA fragments or strands that hybridize and permitpurification of the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker.

The present invention further provides for a pharmaceutical compositioncomprising two or more siRNA molecules for the treatment of any of thediseases and conditions mentioned herein, whereby said two molecules maybe physically mixed together in the pharmaceutical composition inamounts which generate equal or otherwise beneficial activity, or may becovalently or non-covalently bound, or joined together by a nucleic acidlinker of a length ranging from 2-100, preferably 2-50 or 2-30nucleotides. In one embodiment, the siRNA molecules are comprised of adouble-stranded nucleic acid structure as described herein, wherein thetwo siRNA sequences are selected from Tables A, B, C and C2, preferablyfrom Table A, ID Nos: 3, 5, 20 and 23 (human sequences) and 11, 12, 14,17 and 18 (mouse sequences).

In another embodiment, the siRNA molecules are comprised of adouble-stranded nucleic acid structure, wherein the first siRNA sequenceis selected from Tables A, B, C or C2, preferably from Table A, ID Nos:3, 5, 20 and 23 (human p53 sequences) or 11, 12, 14, 17 and 18 (mousep53 sequences) and the second siRNA molecule targets a pro-apoptoticgene, thereby providing beneficial activity. The tandem double-strandedstructure which comprises two or more siRNA sequences is processedintracellularly to form two or more different siRNAs. Such second siRNAmolecule is preferably an siRNA molecule that targets a pro-apoptoticgene.

The siRNA molecules are covalently or non-covalently bound or joined bya linker to form a tandem siRNA molecule. Such tandem siRNA moleculescomprising two siRNA sequences are typically of 38-150 nucleotides inlength, more preferably 38 or 40-60 nucleotides in length, and longeraccordingly if more than two siRNA sequences are included in the tandemmolecule. A longer tandem molecule comprised of two or more longersequences which encode siRNA produced via internal cellular processing,e.g., long dsRNAs, is also envisaged, as is a tandem molecule encodingtwo or more shRNAs. Such tandem molecules are also considered to be apart of the present invention.

siRNA molecules that target p53 may be the main active component in apharmaceutical composition, or may be one active component of apharmaceutical composition containing two or more siRNAs (or moleculeswhich encode or endogenously produce two or more siRNAs, be it a mixtureof molecules or one or more tandem molecules which encode two or moresiRNAs), said pharmaceutical composition further being comprised of oneor more additional siRNA molecule which targets one or more additionalgene. Simultaneous inhibition of p53 and said additional gene(s) willlikely have an additive or synergistic effect for treatment of thediseases disclosed herein.

In a specific example, the pharmaceutical composition for treatment ofthe diseases disclosed herein may be comprised of the following compoundcombinations: 1) p53 siRNA and Fas siRNA; 2) p53 siRNA and Bax siRNA; 3)p53 siRNA and Noxa siRNA; 4) p53 siRNA and Puma siRNA; 5) p53 siRNA andRTP801 siRNA; 6) p53 siRNA and PIDD siRNA; 7) p53 siRNA, Fas siRNA andany of RTP801 siRNA, Bax siRNA, Noxa siRNA or Puma siRNA or PIDD siRNAto form trimers or polymers (i.e., tandem molecules which encode threesiRNAs). Other preferred options of pro-apoptotic genes are siRNAcombinations of any of p53, TNFα, caspase 2, caspase 3, caspase 9, E2F1,and PARP-1. A preferred combination according to the present inventionis p53 siRNA and RTP801 siRNA. (see PCT patent applicationPCT/US2005/029236).

As disclosed herein, aptamers may also be used in the present inventionalone or in combination with the novel siRNAs disclosed herein fortargeting p53 of the invention and for the treatment of any one of theconditions disclosed herein. For example, an aptamer can be used withany one of the siRNAs disclosed herein in combination therapy for thetreatment of any one of the conditions disclosed herein. The novelpharmaceutical composition employed for such a combination therapy,which is also part of the present invention, may comprise an siRNA ofthe present invention covalently or non-covalently attached to anaptamer. Aptamers are RNA or DNA single-strand or double-strandoligonucleic acids which bind to a target protein and do not generallyexhibit non-specific effects. Aptamers can be modified for stability orother desired qualities in accordance with any nucleic acidmodifications disclosed herein and/or known to one of skill in the art.Modifications to aptamers can be introduced anywhere in the molecule,such as the 5′ or 3′ termini, or at any internally defined modificationsite. For example, RNA aptamers can be stabilized with 2′-Fluoro or2′-amino modified pyrimidines. Aptamers can also be linked to reportermolecules or linker chemistries and can be attached to beads or othersolid support if necessary (e.g., 5′ or 3′ amino, thiol ester or biotingroups). Thioaptamers are aptamers which contain sulfur modifications atspecific internucleoside phosphoryl sites, and may possess enhancedstability, nuclease resistance, target affinity and/or selectivity.Examples of thioaptamers include phosphoromonothioate (S-ODN) andphosphorodithioate (S2-ODN) oligodeoxy thioaptamers. For furtherinformation on aptamers and thioaptamers see U.S. Pat. Nos. 5,218,088and 6,423,493.

Additionally, the siRNA disclosed herein or any nucleic acid moleculecomprising or encoding such siRNA can be linked or bound (covalently ornon-covalently) to antibodies (including aptamer molecules) against cellsurface internalizable molecules expressed on the target cells, in orderto achieve enhanced targeting for treatment of the diseases disclosedherein. For example, anti-Fas antibody (preferably a neutralizingantibody) may be combined (covalently or non-covalently) with a p53siRNA molecule. In another example, an aptamer which can act like aligand/antibody may be combined (covalently or non-covalently) with ap53 siRNA molecule.

The term “Covalent bonding” as used herein refers to chemical bondingthat is characterized by the sharing of pairs of electrons betweenatoms.

The term “Noncovalent bonding” as used herein refers to a variety ofinteractions that are not covalent in nature between molecules or partsof molecules that provide force to hold the molecules or parts ofmolecules together, usually in a specific orientation or conformation.These noncovalent interactions include: ionic bonds, hydrophobicinteractions, hydrogen bonds, Van der Waals forces and Dipole-dipolebonds.

The compounds of the present invention can be delivered either directlyor with viral or non-viral vectors. When delivered directly thesequences are generally rendered nuclease resistant. Alternatively thesequences can be incorporated into expression cassettes or constructssuch that the sequence is expressed in the cell as discussed hereinbelow. Generally the construct contains the proper regulatory sequenceor promoter to allow the sequence to be expressed in the targeted cell.Vectors optionally used for delivery of the compounds of the presentinvention are commercially available, and may be modified for thepurpose of delivery of the compounds of the present invention by methodsknown to one of skill in the art.

In one specific embodiment of this invention, topical, intracochlear,transtympanic and transdermal formulations are particularly preferred.They can be administered by subcutaneous injection. Additionally, theycan be administered by implants, in liquid drops to the ear canal,delivered to the scala tympani chamber of the inner ear by transtympanicinjection, or provided as a diffusible member of a cochlear hearingimplant.

A preferred administration mode is directly to the affected portion ofthe ear or vestibule, topically as by implant for example, and,preferably to the affected hair cells or their supporting cells, so asto direct the active molecules to the source and minimize its sideeffects. A preferred administration mode is a topical delivery of thep53 inhibitor(s) onto the round window membrane of the cochlea. Such amethod of administration of other compounds is disclosed for example inTanaka et al. (Hear Res. 2003 March; 177(1-2):21-31).

As noted, the compositions can be injected through chronically implantedcannulas or chronically infused with the help of osmotic minipumps.Subcutaneous pumps are available that deliver active compounds through asmall tubing to the appropriate area. Highly sophisticated pumps can berefilled through the skin and their delivery rate can be set withoutsurgical intervention. Examples of suitable administration protocols anddelivery systems involving a subcutaneous pump device or continuousinfusion through a totally implanted drug delivery system are describedfor example by Harbaugh, J. Neural Transm. Suppl., 24: 271-277 (1987)and DeYebenes et al., Mov. Disord., 2: 143-158 (1987), the disclosuresof which are incorporated herein by reference.

Delivery of therapeutic agents to the inner ear of a subject can be doneby contact with the inner ear or through the external auditory canal andmiddle ear, as by injection or via catheters, or as exemplified in U.S.Pat. No. 5,476,446, which provides a multi-functional apparatusspecifically designed for use in treating and/or diagnosing the innerear of a human subject. The apparatus is capable of deliveringtherapeutic agents into the inner ear or to middle-inner ear interfacetissues. In addition, other systems may be used to deliver the moleculesof the present invention including but not limited to an osmotic pumpwhich is described in Kingma, G. G., et al., “Chronic drug infusion intothe scala tympani of the guinea pig cochlea”, Journal of NeuroscienceMethods, 45:127-134 (1992). An exemplary, commercially-available osmoticpump may be obtained from the Alza Corp. of Palo Alto, Calif. (USA).

It is also envisaged that a long oligonucleotide (typically 25-500nucleotides in length) comprising one or more stem and loop structures,where stem regions comprise the sequences of the oligonucleotides of theinvention, may be delivered in a carrier, preferably a pharmaceuticallyacceptable carrier, and may be processed intracellularly by endogenouscellular complexes (e.g. by DROSHA and DICER as described above) toproduce one or more smaller double stranded oligonucleotides (siRNAs)which are oligonucleotides of the invention. This oligonucleotide can betermed a tandem shRNA construct. It is envisaged that this longoligonucleotide is a single stranded oligonucleotide comprising one ormore stem and loop structures, wherein each stem region comprises asense and corresponding antisense siRNA sequence of an p53 gene. Inparticular, it is envisaged that this oligonucleotide comprises senseand antisense siRNA sequences as depicted in any one of Tables A, B, Cand C2.

As used herein, the term “polypeptide” refers to, in addition to apolypeptide, an oligopeptide, peptide and a full protein.

As used herein, the term “inhibition” of p53 means inhibition of thegene expression (transcription or translation) or polypeptide activity.

Although the inhibitor may be an siRNA molecule, other inhibitorscontemplated to be used in the methods of the invention to inhibit p53and to treat the diseases and conditions described herein are interalia, antisense oligonucleotides, antisense DNA or RNA molecules,proteins, polypeptides and peptides including peptido-mimetics anddominant negatives, and also expression vectors expressing all theabove. Additional inhibitors may be small chemical molecules, whichgenerally have a molecular weight of less than 2000 daltons, morepreferably less than 1000 daltons, even more preferably less than 500daltons. These inhibitors may act as follows: small molecules may affectexpression and/or activity; antibodies may affect activity; all kinds ofantisense may affect the pro-apoptotic gene expression; and dominantnegative polypeptides and peptidomimetics may affect activity;expression vectors may be used inter alia for delivery of antisense ordominant-negative polypeptides or antibodies.

Antisense Molecules

By the term “antisense” (AS) or “antisense fragment” is meant apolynucleotide fragment (comprising either deoxyribonucleotides,ribonucleotides or a mixture of both) having inhibitory antisenseactivity, said activity causing a decrease in the expression of theendogenous genomic copy of the corresponding gene. An AS polynucleotideis a polynucleotide which comprises consecutive nucleotides having asequence of sufficient length and homology to a sequence present withinthe sequence of the target gene to permit hybridization of the AS to thegene. Many reviews have covered the main aspects of antisense (AS)technology and its therapeutic potential (Aboul-Fadl T., Curr Med Chem.2005, 12(19):2193-214; Crooke S T, Curr Mol Med. 2004, 4(5):465-87;Crooke S T, Ann Rev Med. 2004, 55:61-95; Vacek M et al., Cell Mol LifeSci. 2003, 60(5):825-33; Cho-Chung Y S, Arch Pharm Res. 2003,26(3):183-91. There are further reviews on the chemical (Crooke et al.,Hematol Pathol. 1995, 9(2):59-72), cellular (Wagner, Nature. 1994,372(6504):333-5) and therapeutic (Scanlon, et al, FASEB J. 1995,9(13):1288-96) aspects of AS technology. Antisense intervention in theexpression of specific genes can be achieved by the use of modified ASoligonucleotide sequences (for recent reports see Lefebvre-d'Hellencourtet al, 1995; Agrawal, 1996; LevLehman et al, 1997).

AS oligonucleotide sequences may be short sequences of DNA, typically15-30 mer but may be as small as 7-mer (Wagner et al, Nat. Biotech.1996, 14(7):840-4), designed to complement a target mRNA of interest andform an RNA:AS duplex. This duplex formation can prevent processing,splicing, transport or translation of the relevant mRNA. Moreover,certain AS nucleotide sequences can elicit cellular RNase H activitywhen hybridized with their target mRNA, resulting in mRNA degradation(Calabretta et al, Semin Oncol. 1996, 23(1):78-87). In that case, RNaseH will cleave the RNA component of the duplex and can potentiallyrelease the AS to further hybridize with additional molecules of thetarget RNA. An additional mode of action results from the interaction ofAS with genomic DNA to form a triple helix, which can betranscriptionally inactive.

The sequence target segment for the antisense oligonucleotide isselected such that the sequence exhibits suitable energy relatedcharacteristics important for oligonucleotide duplex formation withtheir complementary templates, and shows a low potential forself-dimerization or self-complementation (Anazodo et al., 1996,Biochem. Biophys. Res. Comm. 229:305-309). For example, the computerprogram OLIGO (Primer Analysis Software, Version 3.4), can be used todetermine antisense sequence melting temperature, free energyproperties, and to estimate potential self-dimer formation andself-complimentary properties. The program allows the determination of aqualitative estimation of these two parameters (potential self-dimerformation and self-complimentary) and provides an indication of “nopotential” or “some potential” or “essentially complete potential”.Using this program target segments are generally selected that haveestimates of no potential in these parameters. However, segments can beused that have “some potential” in one of the categories. A balance ofthe parameters is used in the selection as is known in the art. Further,the oligonucleotides are also selected as needed so that analogsubstitution does not substantially affect function.

Phosphorothioate antisense oligonucleotides do not normally showsignificant toxicity at concentrations that are effective and exhibitsufficient pharmacodynamic half-lives in animals (Agrawal, et al., PNASUSA. 1997, 94(6):2620-5) and are nuclease resistant. Antisenseoligonucleotide inhibition of basic fibroblast growth factor (bFGF),having mitogenic and angiogenic properties, suppressed 80% of growth inglioma cells (Morrison, J Biol Chem. 1991 266(2):728-34) in a saturableand specific manner. Being hydrophobic, antisense oligonucleotidesinteract well with phospholipid membranes (Akhter et al., NAR. 1991,19:5551-5559). Following their interaction with the cellular plasmamembrane, they are actively (or passively) transported into living cells(Loke et al., PNAS 1989, 86(10):3474-8), in a saturable mechanismpredicted to involve specific receptors (Yakubov et al., PNAS, 198986(17):6454-58).

Ribozymes

A “ribozyme” is an RNA molecule that possesses RNA catalytic ability(see Cech for review) and cleaves a specific site in a target RNA. Inaccordance with the present invention, ribozymes which cleave mRNA maybe utilized as inhibitors. This may be necessary in cases whereantisense therapy is limited by stoichiometric considerations (Sarver etal., 1990, Gene Regulation and Aids, pp. 305-325). Ribozymes can then beused that will target the a gene associated with a bone marrow disease.The number of RNA molecules that are cleaved by a ribozyme is greaterthan the number predicted by stochiochemistry. (Hampel and Tritz,Biochem. 1989, 28(12):4929-33; Uhlenbeck, Nature. 1987328(6131):596-600).

Ribozymes catalyze the phosphodiester bond cleavage of RNA. Severalribozyme structural families have been identified including Group Iintrons, RNase P, the hepatitis delta virus ribozyme, hammerheadribozymes and the hairpin ribozyme originally derived from the negativestrand of the tobacco ringspot virus satellite RNA (sTRSV) (U.S. Pat.No. 5,225,347). The latter two families are derived from viroids andvirusoids, in which the ribozyme is believed to separate monomers fromoligomers created during rolling circle replication (Symons, 1989 and1992). Hammerhead and hairpin ribozyme motifs are most commonly adaptedfor trans-cleavage of mRNAs for gene therapy (Sullivan, 1994). Ingeneral the ribozyme has a length of from about 30-100 nucleotides.Delivery of ribozymes is similar to that of AS fragments and/or siRNAmolecules.

Screening of Inactivation Compounds for p53:

Some of the compounds and compositions of the present invention may beused in a screening assay for identifying and isolating compounds thatmodulate the activity of a p53 gene, in particular compounds thatmodulate a disorder accompanied by an elevated level of p53 polypeptide.The compounds to be screened comprise inter alia substances such assmall chemical molecules and antisense oligonucleotides.

The inhibitory activity of the compounds of the present invention on p53or binding of the compounds of the present invention to p53 gene may beused to determine the interaction of an additional compound with the p53polypeptide, e.g., if the additional compound competes with theoligonucleotides of the present invention for inhibition of a p53 gene,or if the additional compound rescues said inhibition. The inhibition oractivation can be tested by various means, such as, inter alia, assayingfor the product of the activity of the p53 polypeptide or displacementof binding compound from the p53 polypeptide in radioactive orfluorescent competition assays.

The present invention is illustrated in detail below with reference tothe Examples, but is not to be construed as being limited thereto.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

EXAMPLES General Methods in Molecular Biology

Standard molecular biology techniques known in the art and notspecifically described were generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and as in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and as in Watson et al., Recombinant DNA, ScientificAmerican Books, New York and in Birren et al (eds) Genome Analysis: ALaboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press,New York (1998) and methodology as set forth in U.S. Pat. Nos.4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 andincorporated herein by reference. Polymerase chain reaction (PCR) wascarried out generally as in PCR Protocols: A Guide To Methods AndApplications, Academic Press, San Diego, Calif. (1990). In situ (Incell) PCR in combination with Flow Cytometry can be used for detectionof cells containing specific DNA and mRNA sequences (Testoni et al.,1996, Blood 87:3822.) Methods of performing RT-PCR are also well knownin the art.

Example 1 Generation of Sequences for Active siRNA Compounds

Using proprietary algorithms and the known sequence of gene p53 (SEQ IDNO:1), the sequences of many potential siRNAs were generated. Table Ashows 23 siRNAs which have so far been selected, chemically synthesizedand tested for activity (see Example 2). All these siRNAs are 19-mers.

TABLE A NM_000546 NM_011640 NM_030989 Number Index Sense strandAntisense strand Species (human) (mouse) (rat) 1 Mo3 GUACAUGUGUAAUAGCUCCGGAGCUAUUACACAUGUAC mouse 3 mis 1232-1250 2 mis 2 Hu2′GACUCCAGUGGUAAUCUAC GUAGAUUACCACUGGAGUC human* 1026-1044 3 mis 2 mis 3QHMon1 CAGACCUAUGGAAACUACU AGUAGUUUCCAUAGGUCUG hum, mon 310-328 3 mis 4mis 4 QHMon2 CUACCUCCCGCCAUAAAAA UUUUUAUGGCGGGAGGUAG hum, mon 1378-13961 mis 1 mis 5 QH1 CCCAAGCAAUGGAUGAUUU AAAUCAUCCAUUGCUUGGG human 361-379No No 6 QH2 CCCGGACGAUAUUGAACAA UUGUUCAAUAUCGUCCGGG human 389-407 No No7 QM1 GAGUCACAGUCGGAUAUCA UGAUAUCCGACUGUGACUC mouse No 552-570 2 mis 8QM2 GGAUGUUGAGGAGUUUUUU AAAAAACUCCUCAACAUCC mouse No 680-698 4 mis 9 QM3CAUCUUUUGUCCCUUCUCA UGAGAAGGGACAAAAGAUG mouse 2 mis 808-826 2 mis 10 QM6GGAAUAGGUUGAUAGUUGU ACAACUAUCAACCUAUUCC mouse No 1870-1888 No 11 QM4GGACAGCCAAGUCUGUUAU AUAACAGACUUGCCUGUCC mouse, rat 2 mis 877-895 527-54512 QM5 GAAGAAAAUUUCCGCAAAA UUUUGCGGAAAUUUUCUUC mouse, rat 3 mis1383-1401 1033-1051 13 A17 CUGGGACAGCCAAGUCUGU ACAGACUUGGCUGUCCCAG hum,mus 598-616 874-892 2-mis 14 E2 UCAUCACACUGGAAGACUC GAGUCUUCCAGUGUGAUGAhum, mus, 1012-1030 1288-1306 938-956 rat 15 E6 CACACUGGAAGACUCCAGUACUGGAGUCUUCCAGUGUG hum, mus, 1016-1034 1292-1310 942-960 rat 16 B1GCGCCAUGGCCAUCUACAA UUGUAGAUGGCCAUGGCGC hum 724-742 1000-1018652-668(17) mon, mus 17 B2 CGCCAUGGCCAUCUACAAG CUUGUAGAUGGCCAUGGCG hum,725-743 1001-1019 652-669(18) mon, mus 18 C1 AGUCACAGCACAUGACGGAUCCGUCAUGUGCUGUGACU hum, 745-763 1021-1039 2 mis mon, mus 19 F2UCCGAGUGGAAGGAAAUUU AAAUUUCCUUCCACUCGGA hum, 835-853 1 mis 3 mis mon,dog 20 F3 CCGAGUGGAAGGAAAUUUG CAAAUUUCCUUCCACUCGG hum, 836-854 1 mis 3mis mon, dog 21 G1 GACAGAAACACUUUUCGAC GUCGAAAAGUGUUUCUGUC 873-891 No Nomon, dog 22 H2 GUGUGGUGGUGCCCUAUGA UCAUAGGGCACCACCACAC hum, 895-913 3mis 3 mis mon, dog 23 I5 GAGAAUAUUUCACCCUUCA UGAAGGGUGAAAUAUUCUC hum,1225-1243 2 mis 1 mis mon, dog

Note that in the above Table A, the sense strands of siRNAs 1-23 haveSEQ ID NOS: 3-25 respectively, and the antisense strands of siRNAs 1-23have SEQ ID NOS: 26-48 respectively. siRNA compound No 1 (SEQ ID NOS: 3and 26) is known from the literature (Dirac and Bernards, Reversal ofsenescence in mouse fibroblasts through lentiviral suppression of p53,J. Biol. Chem. (2003) 278:11731) and siRNA No 2 (SEQ ID NOS:4 and 27) isalso known from the literature (Brummelkamp et al. Science 2002,296:550-553). However, the use of these compounds in the methods oftreatment disclosed herein is previously undisclosed and thus novel.

Table B below shows 71 additional 9-mer siRNAs which have been generatedby the proprietary algorithms.

TABLE B gi2689466 gi53575emb gi499622 gi13097806 gbU48957.1 X01237.9dbjAB02 gbBC003596.1 U48957 1MMP53R 0761.1 (Homo (Macaca (Mouse (CanisNo. Source Sense AntiSense sapiens) fascicularis) mRNA) familiaris) 1Human GUACCACCAUCCACUACAA UUGUAGUGGAUGGUGGUAC [806-824] [835-852] 2Human GGAAACUACUUCCUGAAAA UUUUCAGGAAGUAGUUUCC [188-206] [234-247] 3Human AGACUCCAGUGGUAAUCUA UAGAUUACCACUGGAGUCU [894-912] [922-933] 4Human CCAUCCACUACAACUACAU AUGUAGUUGUAGUGGAUGG [812-830] [840-858] 5Human CCACCAUCCACUACAACUA UAGUUGUAGUGGAUGGUGG [809-827] [837-852] 6Human AAACACUUUUCGACAUAGU ACUAUGUCGAAAAGUGUUU [747-765] — 7 HumanCAUGAGCGCUGCUCAGAUA UAUCUGAGCAGCGCUCAUG [655-673] [683-696] 8 HumanCCAUGGCCAUCUACAAGCA UGCUUGUAGAUGGCCAUGG [596-614] [624-640] 9 HumanCCAAGUCUGUGACUUGCAC GUGCAAGUCACAGACUUGG [476-494] — 10 HumanAAACUUUGCUGCCAAAAAA UUUUUUGGCAGCAAAGUUU [2476-2494] — 11 HumanCCCUCCUUCUCCCUUUUUA UAAAAAGGGAGAAGGAGGG [2421-2439] — 12 HumanGCAAGCACAUCUGCAUUUU AAAAUGCAGAUGUGCUUGC [2389-2407] — 13 HumanGGGUCAACAUCUUUUACAU AUGUAAAAGAUGUUGACCC [2367-2385] — 14 HumanGAAGGGUCAACAUCUUUUA UAAAAGAUGUUGACCCUUC [2364-2382] — 15 HumanCUGGAAGGGUCAACAUCUU AAGAUGUUGACCCUUCCAG [2361-2379] — 16 HumanCCAGAGUGCUGGGAUUACA UGUAAUCCCAGCACUCUGG [2321-2339] — 17 HumanGAUGGGGUCUCACAGUGUU AACACUGUGAGACCCCAUC [2249-2267] — 18 HumanGCCAACUUUUGCAUGUUUU AAAACAUGCAAAAGUUGGC [2225-2243] — 19 HumanCCAUGGCCAGCCAACUUUU AAAAGUUGGCUGGCCAUGG [2216-2234] — 20 HumanAGACCCAGGUCCAGAUGAA UUCAUCUGGACCUGGGUCU [288-306] — 21 Human,CCAUCAUCACACUGGAAGA UCUUCCAGUGUGAUGAUGG [878-896] [906-924] mouse 22Human, CAUCACACUGGAAGACUCC GGAGUCUUCCAGUGUGAUG [882-900] [910-928] mouse23 Human, CAUCAUCACACUGGAAGAC GUCUUCCAGUGUGAUGAUG [879-897] [907-925]mouse 24 Human, ACCAUCAUCACACUGGAAG CUUCCAGUGUGAUGAUGGU [877-895][905-923] mouse 25 Human, AUCAUCACACUGGAAGACU AGUCUUCCAGUGUGAUGAU[880-898] [908-926] mouse 26 Human, CACUGGAAGACUCCAGUGGCCACUGGAGUCUUCCAGUG [887-905] [915-933] mouse 27 Human,ACACUGGAAGACUCCAGUG CACUGGAGUCUUCCAGUGU [886-904] [766-784] [914-932]cynomoglus, mouse 28 Human, UCACACUGGAAGACUCCAG CUGGAGUCUUCCAGUGUGA[884-902] [764-782] [912-930] cynomoglus, mouse 29 Human,AUCACACUGGAAGACUCCA UGGAGUCUUCCAGUGUGAU [883-901] [763-781] [911-929]cynomoglus, mouse 30 Human, CACAGCACAUGACGGAGGU ACCUCCGUCAUGUGCUGUG[617-635] [497-515] [645-663] cynomoglus, mouse 31 Human,CACUGGAAGACUCCAGUGG CCACUGGAGUCUUCCAGUG [887-905] [767-785] [915-933]cynomoglus, mouse 32 Human, UCACAGCACAUGACGGAGG CCUCCGUCAUGUGCUGUGA[616-634] [496-514] [644-662] cynomoglus, mouse 33 Human,GUCACAGCACAUGACGGAG CUCCGUCAUGUGCUGUGAC [615-633] [495-513] [643-661]cynomoglus, mouse 34 Human, CCAUCCACUACAACUACAU AUGUAGUUGUAGUGGAUGG[812-830] [692-710] [702-720] cynomoglus, dog 35 Human,CCACCAUCCACUACAACUA UAGUUGUAGUGGAUGGUGG [809-827] [689-707] [699-717]cynomoglus, dog 36 Human, GAAUAUUUCACCCUUCAGA UCUGAAGGGUGAAAUAUUC[1096-1114] [976-994]  [986-1004] cynomoglus, dog 37 Human,CGAGUGGAAGGAAAUUUGC GCAAAUUUCCUUCCACUCG [706-724] [586-604] [596-614]cynomoglus, dog 38 Human, GAGAAUAUUUCACCCUUCA UGAAGGGUGAAAUAUUCUC[1094-1112] [974-992]  [984-1002] cynomoglus, dog 39 Human,CUACAUGUGUAACAGUUCC GGAACUGUUACACAUGUAG [825-843] [705-723] [715-733]cynomoglus, dog 40 Human, AACUACAUGUGUAACAGUU AACUGUUACACAUGUAGUU[823-841] [703-721] [713-731] cynomoglus, dog 41 Human,CAACUACAUGUGUAACAGU ACUGUUACACAUGUAGUUG [822-840] [702-720] [712-730]cynomoglus, dog 42 Human, CACUACAACUACAUGUGUA UACACAUGUAGUUGUAGUG[817-835] [697-715] [707-725] cynomoglus, dog 43 Human,CCACUACAACUACAUGUGU ACACAUGUAGUUGUAGUGG [816-834] [696-714] [706-724]cynomoglus, dog 44 Human, GACAGAAACACUUUUCGAC GUCGAAAAGUGUUUCUGUC[742-760] [622-640] [632-650] cynomoglus, dog 45 Human,GGAGAAUAUUUCACCCUUC GAAGGGUGAAAUAUUCUCC [1093-1111] [973-991] [983-1001] cynomoglus, dog 46 Human, GUGUAACAGUUCCUGCAUGCAUGCAGGAACUGUUACAC [831-849] [711-729] [721-739] cynomoglus, dog 47Human, ACAACUACAUGUGUAACAG CUGUUACACAUGUAGUUGU [821-839] [701-719]]711-729] cynomoglus, dog 48 Human, ACUACAACUACAUGUGUAAUUACACAUGUAGUUGUAGU [818-836] [698-716] [708-726] cynomoglus, dog 49Human, ACCAUCCACUACAACUACA UGUAGUUGUAGUGGAUGGU [811-829] [691-709][701-719] cynomoglus, dog 50 Human, ACCACCAUCCACUACAACUAGUUGUAGUGGAUGGUGGU [808-826] [688-706] [698-716] cynomoglus, dog 51Human, UACCACCAUCCACUACAAC GUUGUAGUGGAUGGUGGUA [807-825] [687-705][697-715] cynomoglus, dog 52 Human, ACAGAAACACUUUUCGACAUGUCGAAAAGUGUUUCUGU [743-761] [623-641] [633-651] cynomoglus, dog 53Human, GAGUGGAAGGAAAUUUGCG CGCAAAUUUCCUUCCACUC [707-725] [587-605][597-615] cynomoglus, dog 54 Human, AUAUUUCACCCUUCAGAUCGAUCUGAAGGGUGAAAUAU [1098-1116] [978-996]  [988-1006] cynomoglus, dog 55Human, AAUAUUUCACCCUUCAGAU AUCUGAAGGGUGAAAUAUU [1097-1115] [977-995] [987-1005] cynomoglus, dog 56 Human, AGAAUAUUUCACCCUUCAGCUGAAGGGUGAAAUAUUCU [1095-1113] [975-993]  [985-1003] cynomoglus, dog 57Human, UGGAGAAUAUUUCACCCUU AAGGGUGAAAUAUUCUCCA [1092-1110] [972-990] [982-1000] cynomoglus, dog 58 Human, ACAUGUGUAACAGUUCCUGCAGGAACUGUUACACAUGU [827-845] [707-725] [717-735] cynomoglus, dog 59Human, UACAACUACAUGUGUAACA UGUUACACAUGUAGUUGUA [820-838] [700-718][710-728] cynomoglus, dog 60 Human, CUACAACUACAUGUGUAACGUUACACAUGUAGUUGUAG [819-837] [699-717] [709-727] cynomoglus, dog 61Human, UCCACUACAACUACAUGUG CACAUGUAGUUGUAGUGGA [815-833] [695-713][705-723] cynomoglus, dog 62 Human, AUCCACUACAACUACAUGUACAUGUAGUUGUAGUGGAU [814-832] [694-712] [704-722] cynomoglus, dog 63Human, CAUCCACUACAACUACAUG CAUGUAGUUGUAGUGGAUG [813-831] [693-711][703-721] cynomoglus, dog 64 Human, CACCAUCCACUACAACUACGUAGUUGUAGUGGAUGGUG [810-828] [690-708] [700-718] cynomoglus, dog 65Human, UGUGUAACAGUUCCUGCAU AUGCAGGAACUGUUACACA [830-848] [710-728][720-738] cynomoglus, dog 66 Human, CAUGUGUAACAGUUCCUGCGCAGGAACUGUUACACAUG [828-846] [708-726] [718-736] cynomoglus, dog 67Human, UACAUGUGUAACAGUUCCU AGGAACUGUUACACAUGUA [826-844] [706-724][716-734] cynomoglus, dog 68 Human, ACUACAUGUGUAACAGUUCGAACUGUUACACAUGUAGU [824-842] [704-722] [714-732] cynomoglus, dog 69Human, AUCCGAGUGGAAGGAAAUU AAUUUCCUUCCACUCGGAU [703-721] [583-601][593-611] cynomoglus, dog 70 Human, UCACUCCAGCCACCUGAAGCUUCAGGUGGCUGGAGUGA [1212-1230] [1092-1110]  [1102-1120] cynomoglus, dog71 Human, CUCACUCCAGCCACCUGAA UUCAGGUGGCUGGAGUGAG [1211-1229][1091-1109]  [1101-1119] cynomoglus, dog

Note that in the above Table B, the sense strands of siRNAs 1-71 haveSEQ ID NOS: 49-119 respectively, and the antisense strands of siRNAs1-71 have SEQ ID NOS: 120-190 respectively.

Table C below shows 63 additional 21-mer siRNAs which have beengenerated by the proprietary algorithms.

Table C2 below shows 35 additional 23-mer siRNAs which have beengenerated by the proprietary algorithms.

TABLE C gi2689466 gi13097806 gbU48957.1 gi53575emb gi4996229 gbBC00359U48957 X01237.1M dbj 6.1 (Macaca MP53R AB020761.1 (Homo fasci- (Mouse(Canis No. Source Sense SIRNA AntiSense SiRNA sapiens) cularis) mRNA)familiaris) 1 Human GGAAGAGAAUCUCCGCAAGAA UUCUUGCGGAGAUUCUCUUCC[975-995] — — — 2 Human GUACCACCAUCCACUACAACU AGUUGUAGUGGAUGGUGGUAC[806-826] [686-706] [835-852] [697-716] 3 Human GGACGAUAUUGAACAAUGGUUAACCAUUGUUCAAUAUCGUCC [261-281] — — — 4 Human CCAGCCACCUGAAGUCCAAAAUUUUGGACUUCAGGUGGCUGG [1217-1237] [1097-1115] — [1107-1120] 5 HumanGAGAAUAUUUCACCCUUCAGA UCUGAAGGGUGAAAUAUUCUC [1094-1114] [974-994][1122-1137] [984-1004] 6 Human AGAAACCACUGGAUGGAGAAUAUUCUCCAUCCAGUGGUUUCU [1079-1099] [959-979] — — 7 HumanCUACUGGGACGOAACAGCUUU AAAGCUGUUCCGUCCCAGUAG [910-930] [790-810] — — 8Human AGACUCCAGUGGUAAUCUACU AGUAGAUUACCACUGGAGUCU [894-914] [774-794][922-933] [784-795] 9 Human CUGGAAGACUCCAGUGGUAAU AUUACCACUGGAGUCUUCCAG[889-909] [769-789] [917-933] [779-795] 10 Human GAAACUACUUCCUGAAAACAAUUGUUUUCAGGAAGUAGUUUC [189-209] [69-87] [235-247] [122-135] 11 HumanGGAAACUACUUCCUGAAAACA UGUUUUCAGGAAGUAGUUUCC [188-208] [68-87] [234-247][122-134] 12 Human AAACACUUUUCGACAUAGUGU ACACUAUGUCGAAAAGUGUUU [747-767][627-647] — [637-657] 13 Human GGAGUAUUUGGAUGACAGAAAUUUCUGUCAUCCAAAUACUCC [729-749] [609-629] — — 14 HumanUCAGACCUAUGGAAACUACUU AAGUAGUUUCCAUAGGUCUGA [178-198] [58-78] [231-244]— 15 Human CCAUGGCCAUCUACAAGCAGU ACUGCUUGUAGAUGCCCAUGG [596-616][476-496] [624-640] [485-495] 16 Human CCAAGUCUGUGACUUGCACGUACGUGCAAGUCACAGACUUGG [476-496] [356-376] — — 17 HumanGGACAGCCAAGUCUGUGACUU AAGUCACAGACUUGGCUGUCC [470-490] [352-370][498-513] [357-377] 18 Human CCCUCCUUCUCCCUUUUUAUA UAUAAAAAGGGAGAAGGAGGG[2421-2441] — [1721-1731] — 19 Human, CCAUCCACUACAACUACAUGUACAUGUAGUUGUAGUGGAUGG [812-832] [692-712] [840-860] [702-722]cynomoglus, dog 20 Human, CCACCAUCCACUACAACUACA UGUAGUUGUAGUGGAUGGUGG[809-829] [689-709] [837-857] [699-719] cynomoglus, dog 21 Human,GAGAAUAUUUCACCCUUCAGA UCUGAAGGGUGAAAUAUUCUC [1094-1114] [974-994] [984-1004] cynomoglus, dog 22 Human, GGAGAAUAUUUCACCCUUCAGCUGAAGGGUGAAAUAUUCUCC [1093-1113] [973-993]  [983-1003] cynomoglus, dog23 Human, CUACAUGUGUAACAGUUCCUG CAGGAACUGUUACACAUGUAG [825-845][705-725] [715-735] cynomoglus, dog 24 Human, ACAACUACAUGUGUAACAGUUAACUGUUACACAUGUAGUUGU [821-841] [701-721] [711-731] cynomoglus, dog 25Human, CCACUACAACUACAUGUGUAA UUACACAUGUAGUUGUAGUGG [8 16-836] [696-716][706-726] cynomoglus, dog 26 Human, CACCAUCCACUACAACUACAUAUGUAGUUGUAGUGGAUGGUG [810-830] [690-710] [700-720] cynomoglus, dog 27Human, GAAUAUUUCACCCUUCAGAUC GAUCUGAAGGGUGAAAUAUUC [1096-11161 [976-996] [986-1006] cynomoglus, dog 28 Human, AGAAUAUUUCACCCUUCAGAUAUCUGAAGGGUGAAAUAUUCU [1095-1115] [975-995]  [985-1005] cynomoglus, dog29 Human, UACCACCAUCCACUACAACUA UAGUUGUAGUGGAUGGUGGUA [807-827][687-707] [697-717] cynomoglus, dog 30 Human, GAUGGAGAAUAUUUCACCCUUAAGGGUGAAAUAUUCUCCAUC [1090-1110] [970-990]  [980-1000] cynomoglus, dog31 Human, CCGAGUGGAAGGAAAUUUGCG CGCAAAUUUCCUUCCAGUCGG [705-725][585-605] [595-615] cynomoglus, dog 32 Human, AACUACAUGUGUAACAGUUCCGGAACUGUUACACAUGUAGUU [823-843] [703-723] [713-733] cynomoglus, dog 33Human, CAACUACAUGUGUAACAGUUC GAACUGUUACACAUGUAGUUG [822-842] [702-722][712-732] cynomoglus, dog 34 Human, ACUACAACUACAUGUGUAACAUGUUACACAUGUAGUUGUAGU [8 18-838] [698-718] [708-728] cynomoglus, dog 35Human, CACUACAACUACAUGUGUAAC GUUACACAUGUAGUUGUAGUG [817-8371 [697-717][707-727] cynomoglus, dog 36 Human, UCCACUACAACUACAUGUGUAUACACAUGUAGUUGUAGUGGA [815-835] [695-715] [705-725] cynomoglus, dog 37Human, CAUCCACUACAACUACAUGUG CACAUGUAGUUGUAGUGGAUG [813-833] [693-713][703-723] cynomoglus, dog 38 Human, ACCAUCCACUACAACUACAUGCAUGUAGUUGUAGUGGAUGGU [811-831] [691-711] [701-721] cynomoglus, dog 39Human, UGGAGAAUAUUUCACCCUUCA UGAAGGGUGAAAUAUUCUCCA [1092-1112] [972-992] [982-1002] cynomoglus, dog 40 Human, AUGUGUAACAGUUCCUGCAUGCAUGCAGGAACUGUUACACAU [829-849] [709-729] [719-739] cynomoglus, dog 41Human, CAUGUGUAACAGUUCCUGCAU AUGCAGGAACUGUUACACAUG [828-848] [708-728][718-738] cynomoglus, dog 42 Human, UACAACUACAUGUGUAACAGUACUGUUACACAUGUAGUUGUA [820-840] [700-720] [710-730] cynomoglus, dog 43Human, CUACAACUACAUGUGUAACAG CUGUUACACAUGUAGUUGUAG [819-839] [699-719][709-729] cynomoglus, dog 44 Human, AUCCACUACAACUACAUGUGUACACAUGUAGUUGUAGUGGAU [814-834] [694-714] [704-724] cynomoglus, dog 45Human, ACCACCAUCCACUACAACUAC GUAGUUGUAGUGGAUGGUGGU [808-828] [688-708][698-718] cynomoglus, dog 46 Human, AAUAUUUCACCCUUCAGAUCCGGAUCUGAAGGGUGAAAUAUU [1097-1117] [977-997]  [987-1007] cynomoglus, dog47 Human, ACUACAUGUGUAACAGUUCCU AGGAACUGUUACACAUGUAGU [824-844][704-724] [714-734] cynomoglus, dog 48 Human, AUGGAGAAUAUUUCACCCUUCGAAGGGUGAAAUAUUCUCCAU [1091-1111] [971-991]  [981-1001] cynomoglus, dog49 Human, UGUGUAACAGUUCCUGCAUGG CCAUGCAGGAACUGUUACACA [830-850][710-730] [720-740] cynomoglus, dog 50 Human, UCCGAGUGGAAGGAAAUUUGCGCAAAUUUCCUUCCACUCGGA [704-724] [584-604] [594-614] cynomoglus, dog 51Human, AUCCGAGUGGAAGGAAAUUUG CAAAUUUCCUUCCACUCGGAU [703-723] [583-603][593-613] cynomoglus, dog 52 Human, UCACACUGGAAGACUCCAGUGCACUGGAGUCUUCCAGUGUGA [884-904] [764-784] [912-932] cynomoglus, mouse 53Human, AUCACACUGGAAGACUCCAGU ACUGGAGUCUUCCAGUGUGAU [883-903] [763-783][911-931] cynomoglus, mouse 54 Human, CACACUGGAAGACUCCAGUGGCCACUGGAGUCUUCCAGUGUG [885-905] [765-785] [913-933] cynomoglus, mouse 55Human, UCAUCACACUGGAAGACUCCA UGGAGUCUUCCAGUGUGAUGA [881-901] [909-929]mouse 56 Human, CCAUCAUCACACUGGAAGACU AGUCUUCCAGUGUGAUGAUGG [878-898][906-926] mouse 57 Human, CAUCACACUGGAAGACUCCAG CUGGAGUCUUCCAGUGUGAUG[882-902] [910-930] mouse 58 Human, CAUCAUCACACUGGAAGACUCGAGUCUUCCAGUGUGAUGAUG [879-899] [907-927] mouse 59 Human,ACCAUCAUCACACUGGAAGAC GUCUUCCAGUGUGAUGAUGGU [877-897] [905-925] mouse 60Human, UCACACUGGAAGACUCCAGUG CACUGGAGUCUUCCAGUGUGA [8 84-904] [912-932]mouse 61 Human, AUCACACUGGAAGACUCCAGU ACUGGAGUCUUCCAGUGUGAU [883-903][911-931] mouse 62 Human, AUCAUCACACUGGAAGACUCC GGAGUCUUCCAGUGUGAUGAU[880-900] [908-928] mouse 63 Human, CACACUGGAAGACUCCAGUGGCCACUGGAGUCUUCCAGUGUG [885-905] [913-933] mouse

TABLE C2 Human- Human- siRNA 120407067; 8400737; name Sense strand ASstrand Other Species ORF:252-1433 ORF:252-1433 1 GGAGAAUAUUUCACCCUUCACAUAUCUGAAGGGUGAAAUAUUCUCC MF, CF 1224-1246 1224-1246 2UGGAGAAUAUUUCACCCUUCAGA UCUGAAGGGUGAAAUAUUCUCCA MF, CF 1223-12451223-1245 3 AUGGAGAAUAUUUCACCCUUCAG CUGAAGGGUGAAAUAUUCUCCAU MF, CF1222-1244 1222-1244 4 GAUGGAGAAUAUUUCACCCUUCA UGAAGGGUGAAAUAUUCUCCAUCMF, CF 1221-1243 1221-1243 5 GAGAAUAUUUCACCCUUCAGAUCGAUCUGAAGGGUGAAAUAUUCUC MF, CF 1225-1247 1225-1247 6AGUGUGGUGGUGCCCUAUGAGCC GGCUCAUAGGGCACCACCACACU MF 894-916 894-916 7UAGUGUGGUGGUGCCCUAUGAGC GCUCAUAGGGCACCACCACACUA MF 893-915 893-915 8AUAGUGUGGUGGUGCCCUAUGAG CUCAUAGGGCACCACCACACUAU MF 892-914 892-914 9CAUAGUGUGGUGGUGCCCUAUGA UCAUAGGGCACCACCACACUAUG MF 891-913 891-913 10GUGUGGUGGUGCCCUAUGAGCCG CGGCUCAUAGGGCACCACCACAC MF 895-917 895-917 11UGACAGAAACACUUUUCGACAUA UAUGUCGAAAAGUGUUUCUGUCA MF, CF, Sus scrofa872-894 872-894 12 AUGACAGAAACACUUUUCGACAU AUGUCGAAAAGUGUUUCUGUCAU MF,CF, Sus scrofa 871-893 871-893 13 GAUGACAGAAACACUUUUCGACAUGUCGAAAAGUGUUUCUGUCAUC MF, CF, Sus scrofa 870-892 870-892 14GGAUGACAGAAACACUUUUCGAC GUCGAAAAGUGUUUCUGUCAUCC MF, CF, Sus scrofa869-891 869-891 15 GACAGAAACACUUUUCGACAUAG CUAUGUCGAAAAGUGUUUCUGUC MF,CF, Sus scrofa 873-895 873-895 16 UCCGAGUGGAAGGAAAUUUGCGUACGCAAAUUUCCUUCCACUCGGA MF, CF 835-857 835-857 17AUCCGAGUGGAAGGAAAUUUGCG CGCAAAUUUCCUUCCACUCGGAU MF, CF 834-856 834-85618 UAUCCGAGUGGAAGGAAAUUUGC GCAAAUUUCCUUCCACUCGGAUA MF, CF 833-855833-855 19 UUAUCCGAGUGGAAGGAAAUUUG CAAAUUUCCUUCCACUCGGAUAA MF, CF832-854 832-854 20 CCGAGUGGAAGGAAAUUUGCGUG CACGCAAAUUUCCUUCCACUCGG MF,CF 836-858 836-858 21 AUCAUCACACUGGAAGACUCCAG CUGGAGUCUUCCAGUGUGAUGAUMus musculus, 1011-1033 1011-1033 O. cuniculus 22CAUCAUCACACUGGAAGACUCCA UGGAGUCUUCCAGUGUGAUGAUG Mus musculus, 1010-10321010-1032 O. cuniculus 23 CCAUCAUCACACUGGAAGACUCCGGAGUCUUCCAGUGUGAUGAUGG Mus musculus, 1009-1032 1009-1032 O. cuniculus24 ACCAUCAUCACACUGGAAGACUC GAGUCUUCCAGUGUGAUGAUGGU Mus musculus,1008-1031 1008-1031 O. cuniculus 25 UCAUCACACUGGAAGACUCCAGUACUGGAGUCUUCCAGUGUGAUGA Mus musculus, 1012-1034 1012-1034 O. cuniculus26 UCCCAAGCAAUGGAUGAUUUGAU AUCAAAUCAUCCAUUGCUUGGGA 360-382 360-382 27GUCCCAAGCAAUGGAUGAUUUGA UCAAAUCAUCCAUUGCUUGGGAC 359-381 359-381 28CGUCCCAAGCAAUGGAUGAUUUG CAAAUCAUCCAUUGCUUGGGACG 358-380 358-380 29CCGUCCCAAGCAAUGGAUGAUUU AAAUCAUCCAUUGCUUGGGACGG 357-379 357-379 30CCCAAGCAAUGGAUGAUUUGAUG CAUCAAAUCAUCCAUUGCUUGGG 361-383 361-383 31UCAGACCUAUGGAAACUACUUCC GGAAGUAGUUUCCAUAGGUCUGA MF 309-331 309-331 32UUCAGACCUAUGGAAACUACUUC GAAGUAGUUUCCAUAGGUCUGAA MF 308-330 30 8-330 33UUUCAGACCUAUGGAAACUACUU AAGUAGUUUCCAUAGGUCUGAAA MF 307-329 307-329 34UUUUCAGACCUAUGGAAACUACU AGUAGUUUCCAUAGGUCUGAAAA MF 306-328 306-328 35CAGACCUAUGGAAACUACUUCCU AGGAACUAGUUUCCAUAGGUCUG MF 310-332 310-332Macaca fascicularis = MF Canis familiaris = CF

Note that in the above Table C, the sense strands of siRNAs 1-63 haveSEQ ID NOS: 191-253 respectively, and the antisense strands of siRNAs1-63 have SEQ ID NOS: 254-316 respectively.

Note that in the above Table C2, the sense strands of siRNAs 1-35 haveSEQ ID NOS: 317-352 respectively, and the antisense strands of siRNAs1-35 have SEQ ID NOS: 353-387 respectively.

Example 2 Testing the siRNA Compounds for Anti-p53 Activity Protocols

I. Preparation of the siRNAs (Double-Stranded Oligonucleotides)

Lyophilized oligonucleotides were dissolved in RNAse free distilledwater to produce a final concentration of 100 uM. The dilutedoligonucleotides were kept at room temperature for 15 min andimmediately frozen in liquid nitrogen.

The oligonucleotides were stored at −80° C. and diluted before use withPBS.

II. Transfection of siRNA in Human Cells with Lipofectamine2000 Reagent:

2×10⁵ p53-wt HCT116 or SW480 cells were seeded per well in 6 wellsplate. 24 h subsequently, cells were transfected with p53oligonucleotides using lipofectamine2000 reagent (obtained fromInvitrogen).

The following procedure was performed:

-   -   1. Before transfection, the cell medium was replaced by 1500 ul        fresh medium without antibiotics.    -   2. In a sterile, plastic tube, Lipofectamine2000 reagent (the        amount is calculated according to 5 ul per well) was added to        250 ul serum-free medium, and incubated for 5 min at room        temperature.    -   3. In another tube the human anti-p53 oligonucleotides (varying        amounts to fit the desired final concentration per well) were        added to 250 ul serum-free medium.    -   4. Lipofectamine2000 complex was combined with the p53        oligonucleotide solution and incubated for 20 min at room        temperature.    -   5. The resulting mixture was added dropwise to the cells, and        the cells were incubated at 37° C.    -   6. SW480 cells: 48 hr after transfection the cells were        harvested and proteins were extracted using RIPA buffer.    -   7. HCT116 cells:        -   40 h after transfection, 5Fu (Sigma) was added to cells to            produce a final concentration of 25 ug/ml. 48 h after cells            transfection (8 h after 5Fu treatment), the cells were            harvested and proteins were extracted using RIPA buffer.    -   8. p53 expression was determined by Western Blot analysis using        monoclonal antibody (Do-1 clone, Santa Cruz). For normalization,        blots were examined for Tubulin expression.        III Co-Transfection of Mouse p53 Gene and Mouse p53        Oligonucleotides into PC3 Cells Using Lipofectamine2000 Reagent:

2×10⁵ p53-null PC3 cells were seeded per well in 6 wells plate. 24 hsubsequently, cells were Co-transfected with mouse p53 gene and GFP geneand mouse p53 oligonucleotides using lipofectamine2000 reagent(Invitrogen). The following procedure was performed:

-   -   1. Before transfection cell medium was replaced by 1500 ul fresh        medium without antibiotics.    -   2. In sterile, plastic tube, Lipofectamine2000 reagent (5 ul per        well) was added to 250 ul serum-free medium, and incubated for 5        min at room temperature.    -   3. In another tube 4 ug DNA (p53 gene:GFPgene, 10:1) and human        p53 oligonucleotides were added to 250 ul serum free medium.    -   4. Lipofectamine2000 complex was combined with p53        oligonucleotides solution and incubated for 20 min at room        temperature.    -   5. The mixture solution was added dropwise to the cells, and        cells were incubated at 37° C.    -   6. 48 h after transfection, cells were harvested and proteins        were extracted using RIPA buffer.    -   7. p53 expression was determined by Western Blot analysis using        monoclonal antibody (Clone240, Chemicon). For normalization,        blots were examined for GFP expression.

Results:

A. Human p53 Oligonucleotides:

TABLE D Results of Test Number oligo species source SW480 HCT116 2 Hu2′human literature (−) (+) 3 QHMon1 human, monkey Proprietary (++) (+++) 4QHMon2 human, monkey Proprietary (−) Not tested 5 QH1 human Proprietary(+++) (+++) 6 QH2 human Proprietary (−) Not tested 13 A17 human, mouseProprietary (−) Not tested 14 E2 human, mouse, rat Proprietary (+) Nottested 15 E6 human, mouse, rat Proprietary (−) Not tested 16 B1 human,mouse, rat Proprietary (−) Not tested 17 B2 human, mouse, ratProprietary (−) Not tested 18 C1 human, monkey, mouse Proprietary (−)Not tested 19 F2 human, monkey, dog Proprietary (−) Not tested 20 F3human, monkey, dog Proprietary (+++) (+++) 21 G1 human, monkey, dogProprietary (+++) Not tested 22 H2 human, monkey, dog Proprietary (+)Not tested 23 I5 human, monkey, dog Proprietary (+++) Not tested Note:The numbers in Table D correspond to the numbers used in Table A, wherethe sense strands of siRNAs 1-23 have SEQ ID NOS: 3-25 respectively, andthe antisense strands of siRNAs 1-23 have SEQ ID NOS: 26-48respectively. As shown in Table D, four human oligonucleotides weretested in two systems SW480 and HCT116, according to Protocols II above.Representative results (Western Blot) on which the Results of Test wasbased are shown in FIG. 3.

TABLE E B. Mouse p53 oligonucleotides: Results of Test PC3 nullcells/exogenous oligo species source mouse p53 1 Mo3 mouse literature(+++) 7 QM1 mouse Proprietary (−) 8 QM2 mouse Proprietary (−) 9 QM3mouse Proprietary (−) 10 QM6 mouse Proprietary (−) 11 QM4 mouse, ratProprietary (+++) 12 QM5 mouse, rat Proprietary (+++) 13 A17 human,mouse Proprietary (−) 14 E2 human, mouse, rat Proprietary (++) 15 E6human, mouse, rat Proprietary (−) 16 B1 human, monkey, mouse Proprietary(−) 17 B2 human, monkey, mouse Proprietary (++) 18 C1 human, monkey,mouse Proprietary (++) 19 G1 human, monkey, dog Proprietary (++) 20 F3human, monkey, dog Proprietary (+++) 21 I5 human, monkey, dogProprietary (−) 22 QHMon1 human, monkey Proprietary (++) Note: Thenumbers in Table E (as for Table D) correspond to the numbers used inTable A, where the sense strands of siRNAs 1-23 have SEQ ID NOS: 3-25respectively, and the antisense strands of siRNAs 1-23 have SEQ ID NOS:26-48 respectively. Representatives of the Western Blot results on whichthe Results of Test was based are shown in FIG. 4.

Example 3 Distribution of Cy3-PTEN siRNA in the Cochlea Following LocalApplication to the Round Window of the Ear

A solution of 1 μg/100 μl of Cy3-PTEN siRNA (total of 0.3-0.4 μg) PBSwas applied to the round window of chinchillas. The Cy3-labelled cellswithin the treated cochlea were analyzed 24-48 hours post siRNA roundwindow application after sacrifice of the chinchillas. The pattern oflabeling within the cochlea was similar following 24 h and 48 h andincludes labeling in the basal turn of cochlea, in the middle turn ofcochlea and in the apical turn of cochlea. Application of Cy3-PTEN siRNAonto scala tympani revealed labelling mainly in the basal turn of thecochlea and the middle turn of the cochlea. The Cy3 signal waspersistence to up to 15 days after the application of the Cy3-PTENsiRNA. These results indicate for the first time that local applicationof siRNA molecules within the round window leads to significantpenetration of the siRNA molecules to the basal, middle and apical turnsof the cochlea.

Example 4 The effect of p53 siRNA Treatment on Carboplatin-Induced HairCell Death in the Cochlea of Chinchilla

Eight Chinchillas were pre-treated by direct administration of p53 siRNAin saline (QM5 molecule in Table A, 1, 10 and 30 μg) to the left ear ofeach animal. Saline was given to the right ear of each animal asplacebo. Two days following the administration of the p53 siRNA, theanimals were treated with carboplatin (75 mg/kg ip). After sacrifice ofthe chinchillas (two weeks post carboplatin treatment) the % of deadcells of inner hair cells (IHC) and outer hair cells (OHC) wascalculated in the left ear (siRNA treated) and in the right ear (salinetreated). Since the effect of the siRNA was similar across dose, thedata was pooled from the 3 doses. As demonstrated in Table F-1 below,carboplatin preferentially damages the inner hair cells in thechinchilla at the 75 mg/kg dose while the outer hair cells remainintact. Furthermore, the p53 siRNA significantly reducescarboplatin-induced inner hair cells loss in the cochlea (53.5% of innerhair cell loss in the p53 siRNA treated cochlea versus 71.9% of innerhair cell loss in the PBS treated cochlea).

TABLE F-1 QM5 siRNA Significantly Reduces Carboplatin-Induced IHC Lossin Chinchilla SIRNA TREATED CONTROL EAR Chinchilla # IHC OHC Chin IHCOHC 8136L 64.7 0.6 8136R 68.8 1.1 8140L 48.2 0.8 8140R 87.6 1.8 8143L53.3 1.5 8143R 64.8 2.4 8149L 38.3 1.9 8149r 68.5 3 8153L 59.7 3.1 8153R58.2 2.1 8197L 50.1 1.2 8197R 61.2 1.5 8200L 45.4 1.7 8200R 82.5 1.58202L 68.5 3.0 8202R 83.5 2.6 Mean Treated 53.5 1.7 Mean Control 71.92.0

Example 5 The effect of p53 siRNA Treatment on Acoustic-Induced HairCell Death in the Cochlea of Chinchilla

The activity of p53 siRNA (QM5) in an acoustic trauma model was studiedin chinchilla. A group of 7 animals underwent the acoustic trauma. Theanimals were exposed to an octave band of noise centered at 4 kHz for2.5 h at 105 dB. The left ear of the noise-exposed chinchillas waspre-treated (48 h before the acoustic trauma) with 30 μg of siRNA in ˜10μL of saline; the right ear was pre-treated with vehicle (saline). Thecompound action potential (CAP) is a convenient and reliableelectrophysiological method for measuring the neural activitytransmitted from the cochlea. The CAP is recorded by placing anelectrode near the base of the cochlea in order to detect the localfield potential that is generated when a sound stimulus, such as clickor tone burst, is abruptly turned on. The functional status of each earwas assessed 2.5 weeks after the acoustic trauma. Specifically, the meanthreshold of the compound action potential recorded from the roundwindow was determined 2.5 weeks after the acoustic trauma in order todetermine if the thresholds in the siRNA-treated ear were lower (better)than the untreated (saline) ear. In addition, the amount of inner andouter hair cell loss was determined in the siRNA-treated and the controlear. FIG. 5 shows the mean threshold results recorded from the roundwindow of siRNA-treated (filled circle) and saline-treated (open circle)chinchillas 2.5 weeks after the acoustic trauma. As demonstrated in FIG.5, the mean thresholds were lower in the siRNA-treated ears versus theuntreated ears. The difference at 4 kHz was statistically significant(p<0.033). These results indicate that p53 siRNA administered to theround window of the cochlea is capable of reducing the damage caused byacoustic trauma.

Table F-2 below shows the loss of outer hair cells (OHC) and inner haircells (IHC) for each animal in the Basal Half of the cochlea (50-100%from the apex). The mean OHC loss in the siRNA-treated ears wassignificantly less than in the control ears (13.9% OHC loss in thesiRNA-treated ear versus 19.6% OHC loss in the control ear, asdetermined by paired t-test). In general, there was less IHC loss thatOHC loss in both siRNA-treated and control ears. The mean IHC loss was4.5% in control ears and 1.3% in the siRNA-treated ears. This differencewas not significant statistically. These results indicate that p53 siRNAadministered to the round window of the cochlea is capable of reducingOHC loss in the Basal Half of the cochlea caused by acoustic trauma.

TABLE F-2 QM5 siRNA Significantly Reduces Acoustic-Induced OHC Loss inthe basal half of cochlea in Chinchilla SiRNA-treated Control (left ear(right ear) Chin# IHC OHC IHC OHC 8146 0.0% 0.1% 6.9% 9.7% 8196 0.7%3.4% 2.5% 13.4% 8220 6.7% 79.7% 19.8% 92.4% 8222 0.0% 2.4% 0.1% 5.0%8237 1.4% 2.3% 1.8% 7.5% 8238 0.1% 3.6% 0.0% 1.7% 8246 0.3% 6.0% 0.6%7.4% Mean 1.3% 13.9% 4.5% 19.6% SD 2.4% 29.1% 7.1% 32.3%

Example 6 The Effect of p53 or 801 siRNA Treatment on Cisplatin-InducedHair Cell Death in the Cochlea of Rats

Male Wistar Rats were tested for basal auditory brainstem response (ABR)thresholds for signals of clicks, 8, 16 and 32 kHz prior to cisplatintreatment. Following the basal auditory brainstem response testing,cisplatin was administered as an intraperitoneal infusion of 13 mg/kgover 30 minutes. Treated ears received either 15 ug/4 microliters of p53siRNA (QM5 molecule in Table A) in PBS or 801 siRNA in PBS (applieddirectly to the round window membrane). The 801 siRNA is designatedREDD14 and has the following nucleotide sequence in the sense strand:5′-GUGCCAACCUGAUGCAGCU-3′ and in the antisense stran:5′-AGCUGCAUCAGGUUGGCAC-3′) Control ears were treated with eithernon-related GFP siRNA or PBS. The siRNA molecules were administeredbetween 3-5 days prior to cisplatin administration in order to permitprotective effect on the cochlea.

The auditory brainstem response (ABR) testing was repeated 3 days aftercisplatin administration. The auditory brainstem response thresholdswere compared between pretreatment and posttreatment and the shift inthresholds is indicated in Table G. Higher shift in thresholds followingcisplatin treatment is indicative for more severe hair cells loss in thecochlea. After the repeat of auditory brainstem response testing,animals were sacrificed and cochleae were removed and processed forscanning electron microscopy (SEM) to quantify outer hair cell (OHC)loss in the hook region (high frequency region). The % outer hair cellloss was calculated by dividing the number of missing or severelydamaged cells by the total number of outer hair cells in the field ofthe photograph.

Table G demonstrates the results obtained from four animals thatunderwent the cisplatin-induced damage and were analysed for outer haircell loss in the Hook region. As revealed from the results, animals thatreceived the siRNA directed against p53 or 801 exhibited lower outerhair cell loss and smaller shifts in the threshold for signals of 32kHz. Both parameters indicate that siRNA directed against the p53 or 801genes (mRNA) is protective against cisplatin-induced damage in thecochlea.

TABLE G Hair cell loss versus threshold shift in cisplatin-treatedcochlea of rats Outer hair Auditory brainstem cell (OHC) response(Threshold Treatment loss shift at 32 KHz) QC/L P53 siRNA (QM5) 50% 10dB QC/R PBS 100%  30 dB QF/L P53 siRNA (QM5) 20% 10 dB QF/R GFP 56% 27.5dB QJ/R 801 siRNA (REDD14) 20% 17.5 dB QJ/L GFP 100%  27.5 dB QN/L 801siRNA (REDD14)  0% 10 dB QN/R PBS 100%  17.5 dB

Example 7 Model Systems of Acute Renal Failure (ARF)

Testing the active siRNA for treating ARF may be done for example byusing sepsis-induced ARF or ischemia-reperfusion-induced ARF

1. Sepsis Induced ARF

Two predictive animal models of sepsis-induced ARF are described byMiyaji T, Hu X, Yuen PS, Muramatsu Y, Iyer S, Hewitt S M, Star R A,2003, Ethyl pyruvate decreases sepsis-induced acute renal failure andmultiple organ damage in aged mice, Kidney Int. November; 64(5):1620-31.These two models are lipopolysaccharide administration and cecalligation puncture in mice, preferably in aged mice.

2. Ischemia-Reperfusion-Induced ARF

This predictive animal model is described by Kelly K J, Plotkin Z,Vulgamott S L, Dagher P C, 2003 January. P53 mediates the apoptoticresponse to GTP depletion after renal ischemia-reperfusion: protectiverole of a p53 inhibitor, J Am Soc Nephrol.; 14(1):128-38.

Ischemia-reperfusion injury was induced in rats following 45 minutesbilateral kidney arterial clamp and subsequent release of the clamp toallow 24 hours of reperfusion. An amount of 250 μg of p53 siRNA (QM5sequence, Table A) was injected into the jugular vein 2 hrs prior to and30 minutes following the clamp. Additional amount of 250 μg of siRNAwere given via the tail vein at 4 and 8 hrs after the clamp. siRNAagainst GFP served as a negative control. The siRNA used in theexperiments described herein had a phosphate group at the 3′ terminus ofboth the sense and antisense strand. The 3′-non-phosphorylated siRNA hasbeen found to have similar biologically activity in an animal model asthe corresponding 3′-phosphorylated siRNA. ARF progression was monitoredby measurement of serum creatinine levels before and 24 hrs postsurgery. At the end of the experiment, the rats were perfused via anindwelling femoral line with warm PBS followed by 4% paraformaldehyde.The left kidneys were removed and stored in 4% paraformaldehyde forsubsequent histological analysis. Acute renal failure is frequentlydefined as an acute increase of the serum creatinine level frombaseline. An increase of at least 0.5 mg per dL or 44.2 μmol per L ofserum creatinine is considered as an indication for acute renal failure.Serum creatinine was measured at time zero before the surgery and at 24hours post ARF surgery.

To study the distribution of p53 siRNA in the rat kidney, Cy3-labeled19-mer blunt-ended siRNA molecules (2 mg/kg) having alternating O-methylmodification in the sugar residues were administered iv for 3-5 min,after which in vivo imaging was conducted using two-photon confocalmicroscopy (not shown). The confocal microscopy analysis revealed thatthe majority of siRNA in the kidneys is concentrated in the endosomalcompartment of proximal tubular cells. Both endosomal and cytoplasmicsiRNA fluorescence were relatively stable during the first 2 hrs postdelivery and disappeared at 24 hrs.

As evident from FIG. 6, there was a ten-fold increase in the level ofserum creatinine following the 45-min of kidney bilateral arterial clamptreatment (PBS treatment). Four injections of p53 siRNA (QM5 sequence,Table A) (2 hrs prior to the clamp and 30 min, 4 h and 8 h after theclamp) significantly reduced the creatinine level in serum by 50%(P<0.001). These results demonstrated that p53 siRNA protected renaltissue from the effects of ischemia-reperfusion injury and thus reducethe severity of ARF.

The effect of p53 siRNA treatment on renal ischemia-reperfusion injurywas further determined by analysing the extent of tubular necrosis inthe renal tissue. Tubular necrosis may be scored as: no damage (damagescoring 0), unicellular, patchy isolated necrosis (damage scoring 1),tubular necrosis in less than 25% of the tissue (damage scoring 2),tubular necrosis in between 25 and 50% of the tissue (damage scoring 3)and tubular necrosis in more than 50% of the tissue (damage scoring 4).FIG. 7 demonstrates the tubular kidney damage expressed as damagescoring (Y-axis) in animals that did not undergo ischemia-reperfusioninjury (group 1) or in ischemia-reperfusion injury animals followingtreatment with either PBS (group 2), two injections of p53 siRNA (group3), three injections of p53 siRNA (group 4) or four injections of p53siRNA (group 5). As revealed by FIG. 7, four injections of p53 siRNA ledto significant decrease in the tubular kidney damage as compared to thePBS control group. FIG. 8 demonstrates that four injections of p53 siRNAtreatment down-regulated the expression of the pro-apoptotic gene Pumain the cortical compartment of the kidney in animal subjected toischemia-reperfusion injury. This indicates that p53 siRNA treatment isinhibited the apoptotic processes in the kidney followingischemia-reperfusion injury.

In an additional set of experiments, the effect of a single siRNAinjection at various time-points pre- and post-clamp was examined. 12mg/kg of p53 siRNA (QM5 sequence, Table A) were injected into thejugular vein either 2 hrs prior to the clamp, 30 minutes prior theclamp, 4 hrs post clamp, 8 hrs post clamp, 12 hrs post clamp or 16 hrspost clamp. The effect of the single injection was calculated as theincrease in serum creatinine level in animals underwent the kidneybilateral arterial clamp with the p53 siRNA treatment compared toanimals underwent the kidney bilateral arterial clamp only. As revealedfrom Table H below, a single injection of p53 siRNA (12 mg/kg) was mosteffective in reducing serum creatinine when administered 4 hourspost-clamp. However, significant effect was observed also when the p53siRNA was administered 2 h or 0.5 h pre-clamp or 8 h post-clamp.Injection of 6 mg/kg of p53 siRNA 4 hrs post clamp was less effectivethan the 12 mg/kg dose, indicating a dose-dependent response of the p53siRNA.

TABLE H The effect of single siRNA injection at various time-points pre-and post- clamp Dosage of siRNA Timing of siRNA single injectionadministration Creatinin levels in mg/dl Clamp only 3.8 mg/dl 12 mg/kg 2h pre-clamp 2.3 mg/dl 12 mg/kg 0.5 h pre-clamp 2.8 mg/dl 12 mg/kg 4 hpost-clamp 1.3 mg/dl 12 mg/kg 8 h post-clamp 2.5 mg/dl 12 mg/kg 12 hpost-clamp 3.3 mg/dl 12 mg/kg 16 h post-clamp 3.4 mg/dl  6 mg/kg 4 hpost-clamp 2.2 mg/dl

The trafficking and degradation characteristics of siRNA molecules inproximal tubular cells of the kidney was also analyzed. Systemicallyinfused Cy-3 labeled siRNA was quickly filtered, bound to the apicalbrush border of proximal tubule cells, and internalized into lysosomes60 minutes post infusion. An increase in the fluorescence of thelysosomal pool occurs up to 2 hours post infusion followed by a drop offat the next observed timepoint of 4 hours. The cytosolic componentremains constant throughout the initial timepoints up to 2 hours,suggesting a degradation process occurs once this pool enters thecytosol.

1. A method of treating a patient suffering from a kidney injuryfollowing an ischemic-reperfusion event comprising administering to thepatient a composition comprising one or more compounds having thestructure: 5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y) 5′ (sensestrand) wherein each of N and N′ is a ribonucleotide which may bemodified or unmodified in its sugar residue; wherein each of (N)_(x) and(N′)_(y) is an oligomer in which each consecutive N or N′ is joined tothe next N or N′ by a covalent bond; wherein each of x and y is aninteger between 19 and 40; wherein each of Z and Z′ may be present orabsent, but if present is 1-5 consecutive nucleotides covalentlyattached at the 3′ terminus of the strand in which it is present;wherein the sequence of (N)_(x) comprises an antisense sequence to mRNAof p53; and wherein the composition is administered in a therapeuticallyeffective dose following the initiation of the ischemic-reperfusionevent so as to thereby treat the patient.
 2. The method of claim 1,wherein each of (N)_(x) and (N′)_(y) is set forth in any one of SEQ IDNOS: 3-387.
 3. The method of claim 1, wherein the ischemic-reperfusionevent is a result of a major surgery.
 4. The method of claim 3, whereinthe major surgery is cardiac surgery.
 5. The method of claim 1, whereinthe kidney injury is acute renal failure.
 6. The method of claim 1,wherein the composition is administered between 2-8 hours following theinitiation of the ischemic-reperfusion event.
 7. The method of claim 1,wherein the patient is a high-risk patient undergoing major surgery. 8.The method of claim 6, wherein the composition is administered 4 hoursfollowing the initiation of the ischemic-reperfusion event.
 9. Apharmaceutical composition comprising one or more compounds of claim 1or a vector capable of expressing such compounds in an amount effectiveto treating a patient suffering from ischemic-reperfusion-related kidneyinjury, and a pharmaceutically acceptable carrier.
 10. The compositionof claim 9, wherein the compound having an antisense sequence set forthin serial number 23 in Table A (SEQ ID NO: 25).
 11. A method for theprevention of a kidney injury following an ischemic-reperfusion event ina high-risk patient undergoing major surgery comprising administering tothe patient a composition comprising one or more compounds of claim 2,wherein the composition is administered in a therapeutically effectivedose following the initiation of the ischemic-reperfusion event so as tothereby prevent the kidney injury.
 12. The method of claim 11, whereinthe ischemic-reperfusion event is the result of removal of thecardiopulmonary bypass machine.
 13. A method for the prevention of akidney injury following an ischemic-reperfusion event in a high-riskpatient undergoing major surgery comprising administering to the patienta composition comprising one or more siRNA compounds which have a strandcomplementary to p53 mRNA, wherein the composition is administered in atherapeutically effective dose following the initiation of theischemic-reperfusion event so as to thereby prevent the kidney injury.14. A compounds having the structure: 5′ (N)_(x)-Z 3′ (antisense strand)3′ Z′-(N′)_(y) 5′ (sense strand) wherein each of N and N′ is aribonucleotide which may be modified or unmodified in its sugar residue;wherein each of (N)_(x) and (N′)_(y) is an oligomer in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein each of x and y is an integer between 19 and 40; wherein each ofZ and Z′ may be present or absent, but if present is 1-5 consecutivenucleotides covalently attached at the 3′ terminus of the strand inwhich it is present; wherein each of (N)_(x) and (N′)_(y) is set forthin any one of SEQ ID NOS: 317-387.